Transcript Slide 1

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Cells
Microscopes
Light
Atoms
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Cell structure
Electromagnetic radiation
Atomic models
Fluid transport
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Messenger-mediated calcium signaling
Cell microinjection
Carbon nanotubes
Binding energy
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Carbon nanopipettes
IP3, NAADP, cADPr
Hagen-Poiseuille
Stokes’ shift
MG Schrlau
CELL NANOSURGERY:
Delivering Material into Cells
and Analyzing Effects
ITEST Content Module
Michael G. Schrlau
Mechanical Engineering and
Applied Mechanics
University of Pennsylvania
About Me - Background
University of Pittsburgh
(BSME, 1998)
Elizabeth Jean
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MG Schrlau
About Me - Background
Kimberly-Clark (1998-2004)
• Big-people diapers
• Little-people diapers
• Tissue Paper
University of Pennsylvania (PhD, 2004-Dec
2008 (Expected))
• Nanotechnology Research Nanoprobes
• Nanotechnology Instructor for the
Summer Academy for Applied Science
and Technology (SAAST, 2005)
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MG Schrlau
About Me – Research Interests
Carbon Nanopipette
Carbon Tip
(1) Development and
Fabrication of Nanoprobes
• Intracellular probes
• Nanoelectrodes
• Magnetic probes
5 μm
Quartz Micropipette
MG Schrlau et al, Nanotechnology (2008a and b)
(2) Application of Nanoprobes
• Intracellular material
delivery and
manipulation
• Electrochemical
detection and sensing
HH Bau and MG Schrlau, U.S. Patent Appl. No. 60/888,375
MG Schrlau, Unpublished (2008)
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MG Schrlau
About Me – Research Interests
Carbon Nanopipette
(3) Nanoscale Characterization
• Optical microscopes
• Electron microscopes
(Scanning and Transmission)
(4) Cell Physiology
• Intracellular signaling
• Electrophysiology
• Fluorescence
• Microinjection
SEM
TEM
HRTEM
MG Schrlau et al, Nanotechnology (2008a)
Intracellular Calcium Signaling
Targeting
Before Injection
After Injection
MG Schrlau et al, Nanotechnology (2008b)
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MG Schrlau
Module Topics
Nanosurgery - Using nanoprobes to deliver
material into single cells and analyzing their
response.
Including:
• An overview of cells, intracellular components,
and their functions
• Delivering material into cells - microinjection
• Fluid transport through nanoscale channels
• Visualizing material transport and cellular
response
• Light and optical microscopes
• Molecules and fluorescence
• An example using Carbon Nanopipettes
(CNPs)
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MG Schrlau
Fitting the Topics into the High School Curriculum
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An overview of cells, intracellular components, and their functions
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G10: Biology: Unit 3: Cell Structure and Function
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Delivering material into cells – microinjection
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G9: Phys Sci: Unit 6: Forces & Fluids
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Cell Theory
Techniques of microscope use
Cell organelles – membrane, ER, lysosomes
Fluid pressure
Fluid transport through nanoscale channels
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G9: Phys Sci: Unit 6: Forces & Fluids
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Fluid pressure
G9: Phys Sci: Unit 11: Matter
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Classifying matter
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MG Schrlau
Fitting the Topics into the High School Curriculum
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Visualizing material transport and cellular response
• Light and optical microscopes
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G10: Biology: Unit 3: Cell Structure and Function
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Techniques of microscope use
G9: Phys Sci: Unit 10: Waves
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Electromagnetic waves
Optics
• Molecules and fluorescence
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G10: Biology: Unit 2: Introduction to Chemistry
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G10: Biology: Unit 3: Cell Structure and Function
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Chemistry of water
Techniques of microscope use
G9: Phys Sci: Unit 12: Atoms and the Periodic Table
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Historical development of the atom
Modern atomic theory
Mendeleyev’s periodic table
Modern periodic table
• An example using Carbon Nanopipettes (CNPs)
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MG Schrlau
Introduction into Nanosurgery
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MG Schrlau
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Courtesy of DOE: www.nano.gov/html/facts/The_scale_of_things.html
MG Schrlau
Cells are the building blocks of life
Smallest living unit that can perform functions of life
• Metabolism
• Material Transport
• Reproduction / growth - mitosis
All living things contain cells
10-100 trillion cells in the adult human body
Over 200 hundred different cells / functions
Need microscopes and nanoscale tools to work with cells
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MG Schrlau
The Importance of Cells
Many different types of cell in the human body
To know how cells work is to know how the body
works.
“Cell Nanosurgery” is a way to probe single cell
environments and repair/replace/modify
intracellular components.
http://www.dryggirl.com/teaching/art1/figures/vitruvian_man.jpg
Stem cell
Neuron cell
White & red
blood cells
www.immediart.com/catalog/images/
big_images/SPL_ItB_P360276-Granule
_nerve_cell,_SEM.jpg
bp0.blogger.com
http://epigenome.eu/media/images/large/34.jpg
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Bone cells
www.itg.uiuc.edu/technology
/reconstruction/sem-cells.jpg
MG Schrlau
Why Nanosurgery on Cells?
(1) Fundamental Cell Biology
• What does a cell contain?
Ex: organelles, proteins
• What kind of cell processes
take place?
Ex: cell division
• How does a cell know when to
do particular tasks?
Ex: cell cycles
• How does a cell react to
outside stimulus?
Ex: drugs
www.imgenex.com/emarketing/091406_Glutamate/glutamatepathway.jpg
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MG Schrlau
Why Nanosurgery on Cells?
(2) Nanomedicine – repairing subcellular components or processes
Ex: Gene Therapy
http://www.nwbio.com/images/one_educated.gif
http://www.nwbio.com/images/dcvax_process.gif
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MG Schrlau
Performing Surgery on the Macroscale
Surgery or “hand work” is the physical intervention to investigate a process
or problem and/or repair, replace, or modify a part of the body.
www.melomed.co.za
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MG Schrlau
Areas in Macroscale Surgery
Material
Delivery
Cutting
www. jupiterimages.com
www.south-norfolk.gov.uk
Macroscale Surgery
Sensing
Manipulating
www. jupiterimages.com
www.altramedical.com
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MG Schrlau
Sizing-Down Surgery
Investigate a problem or repair-replace-modify a component
3m
Cells
100 μm
60μm
Nucleus, Organelles
10 μm
1 μm
Proteins, Cytoskeleton, DNA
100 nm
10 nm
1 nm
Surgery can be performed on cells using tools with nanoscale resolution
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MG Schrlau
Developing Areas of Cell Nanosurgery
Material
Delivery
Cutting
Schrlau (Unpublished)
Shen et al (MCB, 2005)
Cell Nanosurgery
Kim and Lieber (Science, 1999)
Sensing
Manipulating
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Yum (ACSNano, 2007)
MG Schrlau
How is Material Delivered into Cells?
• Variety of Techniques
• Viral
• Non-viral
• Chemical endocytosis
• Phagocytosis of Particles
• Injection of Fluids
MG Schrlau, 2008, unpublished
• Fluid Delivery
• Through nanochannels
• Minimally invasive to cells
• Minimal damage to cells
www.gamasutraexchange.com/FullPreview/Index.cfm/ID/2141
04/intType/7/stgCHSource/Popular
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MG Schrlau
Why Deliver Materials into Cells?
1) Permanently change or alter cell
behavior – stem cell
differentiation
Ex: Modify a cell so that it
internally produces and
expresses a green fluorescent
protein (GFP)
http://www.st-andrews.ac.uk/~icmp/Research/research.html
Nucleus produces
RNA-GFP
Cell produces GFP
Deliver DNAGFP Plasmid
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MG Schrlau
Why Deliver Materials into Cells?
2) Investigate response to stimulus
Ex: Determine if a cell releases
calcium in the presence of a
molecule.
MG Schrlau, 2008, unpublished
Molecule binds to
some organelle
Organelle releases
calcium
Deliver molecule
?
?
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MG Schrlau
Delivering Microscopic Material to Cells
ES Cells (~15 μm diameter) can be
easily resolved with visible light
(400-700nm)
Mouse embryos (4 day blastocyst)
injected with embryonic stem (ES)
cells
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MG Schrlau
Delivering Microscopic Material to Cells
Oral Cancer Cell (~15 um
diameter) injected with
fluorescent protein (few nm)
Proteins can not be resolved
with visible light so
fluorescence is used
MG Schrlau, 2008, unpublished
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MG Schrlau
Injection-Mediated Intracellular Calcium Signaling
Inverted Microscope (Nikon)
Manipulator
(Eppendorf)
Perfusion System
Filter Wheel
(Sutter)
Injection System
(Eppendorf)
CCD Camera (Roper)
Ex
Em
Breast cancer cells
(SKBR3) loaded
with Fura-2AM
Ex: 340, 380 nm
Em: 540 nm
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Fluorescent Images (340/380)
Basal
Release
MG Schrlau
Module Topics
Nanosurgery - Using nanoprobes to deliver
material into single cells and analyzing their
response.
Including:
• An overview of cells, intracellular components,
and their functions
• Delivering material into cells - microinjection
• Fluid transport through nanoscale channels
• Visualizing material transport and cellular
response
• Light and optical microscopes
• Molecules and fluorescence
• An example using Carbon Nanopipettes
(CNPs)
‹#›
MG Schrlau
An Overview of Cells, Intracellular
Components, and Their Functions
G10: Biology: Unit 3: Cell Structure and Function
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MG Schrlau
What is a Cell?
Cells are the building blocks of life
All living things contain cells
Smallest living unit that can perform
functions of life
• Metabolism
• Material Transport
• Reproduction / growth - mitosis
10-100 trillion cells in the adult human
body
Over 200 hundred different cells /
functions
http://www.dryggirl.com/teaching/art1/figures/vitruvian_man.jpg
Fun Fact - Connected together, the cells in the body would stretch around
earth 47 times
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MG Schrlau
Anatomy of a Cell
http://people.eku.edu/ritchisong/RITCHISO/301notes1.htm
= Membrane-bound organelles
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MG Schrlau
Anatomy of a Cell
Molecular Biology of the Cell, 4th Edition
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MG Schrlau
Plasma Membrane
Lipids are amphiphilic
• Likes water (hydrophilic)
• Dislikes water (hydrophobic)
Permeability barriers - water-soluble solutes
cannot pass freely across the lipid bilayer
Anchoring cells to surfaces
Very flexible; large deflections
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MG Schrlau
Cytoplasm
Consists of:
• Cytosol
• Organelles
• Cytoskeleton
• Very viscous
(10-100K times
more viscous than
water!)
Cytosol
http://people.eku.edu/ritchisong/RITCHISO/301notes1.htm
• Makes-up majority of cell
• Translucent concoction of water, salts, and organic material
• Space for material and signal transport
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MG Schrlau
Cytoskeleton
Function:
• Cell mobility and
strength
• Material transport
Consists of:
• Microtubules
(yellow, 25nm)
• Microfilaments (blue,
actin, 8nm)
• Intermediate
Filaments (10nm)
M.G. Schrlau UPenn, 2008.04.11
www.immediart.com/catalog/product_info.php?cPath=61_78&products_id=456
Responsible for most
viscosity of cytoplasm
Nucleus
Storage of genetic
information (DNA)
Transports material through
pores
Epithelial Cell
http://cellbio.utmb.edu/cellbio/nucleus.htm
www.itg.uiuc.edu/technology/atlas/structures/nucleus/
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MG Schrlau
Endoplasmic Reticulum (ER)
Function:
• Protein synthesis, folding,
and transport
• Calcium signaling
Complex maze of tubules
Network extends throughout cell
Ribosomes – protein synthesis
Courtesy of Dun Lab, Temple University
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MG Schrlau
Other Organelles
Golgi - packages and
transports material from ER
to specific cell sites,
Involved in the creation of
Lysosomes
http://www.lifesci.sussex.ac.uk/home/Julian_Thorpe/golgi.htm
Lysosomes –
Intracellular
digestion, calcium
signaling
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MG Schrlau
Other Organelles
Centrosome – organization of microtubule network
Vacuole – intracellular digestion, isolation of waste and harmful material
Mitochondrion – power generator of a cell
Mitochondrion
Centrosome
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Mitchison.gif
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MG Schrlau
Anatomy of a Cell
http://people.eku.edu/ritchisong/RITCHISO/301notes1.htm
Cell components work together to perform a variety of functions:
Ex: Intracellular calcium signaling
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MG Schrlau
Intracellular Calcium Signaling
Intracellular Ca+2 regulates processes by activating or inhibiting signaling
pathways or proteins
Long Term
• Gene expression
• Cell cycles
• Growth
• Division
• Apoptosis
Short Term
• Secretion
• Contraction
• Synaptic
transmission
• Metabolism
Second messengers transduce certain membrane signals to release stored
calcium from intracellular stores
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MG Schrlau
Why Study 2nd Messengers & Calcium Signaling?
Unregulated calcium release
implicated in cancer – only IP3
has been studied
(Monteith et al, Nat Rev Cancer, 2007)
http://people.eku.edu/ritchisong/RITCHISO/301notes1.htm
Some Second Messengers:
• IP3 – Inositol triphosphate
• cADPr – Cyclic adenosine diphosphate ribose
• NAADP – Nicotinic acid adenine dinucleotide phosphate
Calcium Stores:
• Endoplasmic Reticulum (ER) – sensitive to IP3 and cADPr (in some cells)
• Lysosomes (Ly) – sensitive to NAADP**
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MG Schrlau
In Short, Cells are Complex!
http://www.medifast1.com/shopping/images/products/stew_large.jpg
http://people.eku.edu/ritchisong/RITCHISO/301notes1.htm
Cells are crowded environments
housing a variety of organelles
and skeletal structures separated
by an aqueous fluid containing
salt and organic material.
www.daylife.com/photo/0fcw2UV6LU9Hq
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MG Schrlau
Additional Reading & References
Interactive cell model
• www.cellsalive.com/cells/cell_model.htm
HyperLink
Cell Biology Text
• Alberts, Molecular Biology of the Cell, 4th Edition, Garland Science, 2002
Intracellular calcium manuscripts
• Monteith et al, Nat Rev Cancer, 2007
• Carafoli et al, Crit Rev Biochem Mol Biol, 2001
• Galione, Biochem Soc Trans, 2006
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MG Schrlau
Delivering Material into Cells
G9: Phys Sci: Unit 6: Forces & Fluids
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MG Schrlau
Obstacles for Material Delivery
Cells are crowded environments housing a variety of organelles
and skeletal structures separated by an aqueous fluid containing
salt and organic material.
Cell are protected by a lipid membrane barrier that controls what
moves across.
‹#›
MG Schrlau
Getting Things into Cells is Challenging
 Intracellular environment is different from the
inside and outside
 Cells are hardy but also very fragile - sensitive
to membrane damage and changes in ion
contents
 Foreign objects can cause inflammatory
response
Intracellular
Extracellular
150mM K+
4mM K+
20mM Na+
145mM Na+
4mM Cl-
110mM Cl-
0.1μM Ca++
2mM Ca++
 Cell is crowded space – organelles could be
damaged or destroyed
Goals of delivering material to cells
• Don’t kill the cell outright
• Don’t damage the cell so that it can’t recover
• Don’t adversely change the cell  unwanted increases in intracellular calcium
• controllably deliver the material to the cell
• Delivery vector can’t be toxic (to cell or organism)
• Safe and efficient
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MG Schrlau
Some Methods of Delivering Material into Cells
• Viral transfection
• Non-Viral Transfection:
Liposomes
Phagocytosis of nanoparticles
Electroporation
Phototransfection (Laser ablation)
Delivering nanoparticles to cell with a probe
Injection of fluids
‹#›
MG Schrlau
Viral Transduction (or Infection)
Using viruses to modify cells by delivering DNA
http://fig.cox.miami.edu/~cmallery/150/gene/sf11x1virus.jpg
1)
Introduce virus
4)
2)
3)
Plate of cells
Cells
Virus
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Infected cells
MG Schrlau
Viral Transduction (or Infection)
Advantages - Very efficient, Can modify many cells (>thousands)
Disadvantages - Safety concerns, can’t delivery drugs
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MG Schrlau
Non-Viral Transfection
Sometimes called Physical transfection – delivering the molecule, drug,
protein, etc. directly to the cell by some physical means.
Types of non-viral transfection
• Material contained inside a vesicle
• Material attached to particle surface
• Material delivered directly be a probe
Advantages
• Eliminates safety concerns
• A variety of techniques to choose from for specific applications
• Can be used for large groups of cells or individual cells
Disadvantages
• Less efficient than viral transduction
• Technically demanding
• No best method for all applications
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MG Schrlau
Non-Viral Transfection
Liposomes
• Encapsulate a sample inside
a bilayer liposome
• Capsule makes contact with
the cell membrane
• Contents are released
www.bio.davidson.edu/courses/GENOMICS/method/liposome.html
Charged copolymers
• DNA binds to polymer particle
• Particle binds to cell
• Cell brings in particle (endocytosis)
www.nano-lifescience.com/images/dna-transport.gif
• Cell populations, technically undemanding transfection
• No control of concentration or delivery location
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MG Schrlau
Non-Viral Transfection
Phagocytosis
• Solid material comes in contact with
cell (gravity, centrifuge, magnet)
Cell brings in the solid particle
Ex: Magnetofection
www.chemicell.com/products/magnetofection/_img/magneto1.jpg
http://img.tfd.com/dorland/thumbs/phagocytosis.jpg
MG Schrlau & B Polyak, Unpublished, 2008
• Cell populations, technically undemanding transfection
• No control of concentration or delivery location, particles left in cell
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MG Schrlau
Non-Viral Transfection
Projectile Delivery
Ex: Magnetic Spearing
• Magnetic projectiles
loaded with material are
pulled toward cells with
a magnet
Cai Nature 2005
Cai Nature 2005
• Cell populations, technically undemanding transfection
• No control of concentration or delivery location, projectile left in cell
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MG Schrlau
Non-Viral Transfection
Projectile Delivery
Gene Gun
http://fig.cox.miami.edu/~cmallery/150/gene/38x15dnagun.jpg
www.bio.davidson.edu/COURSES/Bio111/genegun.html
• Cell populations, technically undemanding transfection
• No control of concentration or delivery location, particles left in cell
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MG Schrlau
Non-Viral Transfection for Single Cells
Makes membrane permeable
(presumably new holes) to
external molecules.
Single-Cell Electroporation
Olofsson et al, Curr Opin Biotechnol, 2003
• Cell populations or single cells
• No control of concentration, semi-elaborate transfection setup
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MG Schrlau
Non-Viral Transfection for Single Cells
Photoporation (Laser Ablation)
Burn a hole in the
membrane with a laser
and let material diffuse in
Larger Pipette with
DNA in fluid
Laser
Cell on cover slip
• Single select cells, excellent position control, eliminate physical probes
• No control of concentration, technically demanding transfection,
diffusion of extracellular material into cells
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MG Schrlau
Non-Viral Transfection for Single Cells
Needle-like Nanoprobes
Chen et al (PNAS, 2007)
• Single select cells, excellent position control
• Limited control of concentration, technically demanding transfection,
difficult probe manufacturing, requires investment in AFM
‹#›
MG Schrlau
Non-Viral Transfection for Single Cells
Fluid Microinjection – most widely-used technique for single-cell transfection
Hollow Nanoprobe – Nanochannel connected to microscopic channel
Submicron Diameter
EXTRACELLULAR
Cylindrical Nanochannel
Fluid inside
INTRACELLULAR
Cell Membrane
Nanochannel
connected to
larger probe
• Single select cells, excellent position control, quick controlled delivery,
easy probe fabrication, fluid delivery
• Technically demanding transfection
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MG Schrlau
The Many Ways of Delivering Material into Cells
Viral transfection
• Very efficient
• Safety concerns
Liposomes
Charged
Copolymers
Non-Viral Transfection:
• Eliminates safety concerns
• Not as efficient
Nanoparticles
Nanoprobes
Focus on fluid microinjection
‹#›
MG Schrlau
Additional Reading & References
Manuscripts
• Leary et al, Neurosurgery, 2005-2006 – reviews nanosurgery and
various approaches
• Stephens and Pepperkok, PNAS, 2001 – reviews different ways of
getting into cells
• Chen et al, PNAS, 2007 – need-like carbon nanoprobes
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MG Schrlau
Fluid Transport Through Nanoscale Channels
G9: Phys Sci: Unit 6: Forces & Fluids
G9: Phys Sci: Unit 11: Matter
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MG Schrlau
Injection of Fluids
Glass Micropipettes
Submicron Diameter
www.eppendorfna.com
Advantages
Disadvantages
• Widely-used, most trusted
• Platform technology for modern
cell physiology
•Relatively easy to make
•Low cost
•
•
•
•
•
‹#›
Single function
Fragile
Large for nanosurgery
Invasive
Can cause irreparable damage to
cell membrane
MG Schrlau
Microinjection Through Nanochannels
Concept of Microinjection
EXTRACELLULAR
Cylindrical Nanochannel
INTRACELLULAR
Fluid inside
Cell Membrane
Nanochannel
connected to
larger probe
Simplified Model of Microinjection
P1
Pinjection
D
Flow
P2
L
Flow
P1 > P2
‹#›
MG Schrlau
Carbon Nanotube and Nanopipes
Carbon Nanotubes
Carbon Nanopipes
Whitby and Quirke
(Nat. Nanotech, 2007)
Minimally invasive probes for
material delivery and sensing
•
•
•
•
Iijima (Nature, 1991)
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High aspect ratio
Nanoscopic channels
High mechanical strength
High electrical conductivity
MG Schrlau
Carbon Nanotubes and Carbon Nanopipes
Carbon nanotubes (CNTs)
• First discovered by Iijima (1991)
Carbon Nanotubes
Iijima (Nature, 1991)
Single Wall
(SWCNT)
Multi Wall
(MWCNT)
Copyright 2000 Scientific American, Inc.From Nanotube for electronics
Configuration of SWNT dictates whether
it’s metallic or a semiconductor.
Nanotubes and Nanofibers, Y Gogotsi
(Ed.), CRC Press, 2006
www.geocities.com/nu_fermi1/swnt.gif
‹#›
www.reading.ac.uk/Physics/
pgprogrammes/MSCprojects2008.htm
MG Schrlau
Carbon Nanotubes and Carbon Nanopipes
SWCNT - Diameters of 0.6 to 1.8 nm,
lengths of 20 nm to 500 mm, high strength,
electrical and thermal conductivity
MWCNT - Diameters >0.6 nm, lengths of 20
nm to 500+ mm, high strength, electrical and
thermal conductivity
MWCNT
10 nm
www.nano-lab.com/nanotube-image3.html
Carbon Nanopipes
• Diameters >50nm, lengths of 20 nm to 500+
mm, high strength, electrical and thermal
conductivity
Rossi et al, Nano Lett. (2004)
• Amorphous carbon but can be annealed at
high temperatures to become more graphitic
‹#›
MG Schrlau
Pushing Fluids Through Channels
How do we deliver liquids to cells with carbon
nanotubes and nanopipes?
• Electroosmosis – movement of fluid under
an applied electric field
• Pressure driven flow – movement of fluid as
a result of a pressure gradient (>50nm)
Gogotsi et al, App. Phys. Lett. (2001)
Courtesy of E Vitol, Gogotsi Group, Drexel University, 2008
www.borealisgroup.com/images/infrastructure/pipes-fittings/PE100_water_pipe.jpg
‹#›
MG Schrlau
Pressure Injection Systems
Eppendorf Femtojet Injector
Narishige Piston Injector
www.narishige.co.jp/products/group1/im-9b.htm
www.eppendorfna.com
• Pressure is set and pulse
time controls volume
• Piston displacement controls
pressure and volume
• Capable of 6000 hPa
‹#›
MG Schrlau
Pushing Fluids Through Channels
Lv 
Reynolds Number (Re):
Inertial Forces
Re 

m
Viscous Forces
L  length (m)
v  velocity (m / s )
  density (kg / m3 )
m  vis cos ity (kg / m  s )
Re>>1:
Re=1:
Re<<1:
Inertia dominates Turbulence
Equal contribution
Viscosity dominates  Laminar
**In micro/nano environments, typically Re<<1
‹#›
MG Schrlau
Determining Flow Through a Pipe
2D Flow Profile
Flow (Q), P1 > P2
P1
μ
D
P2
L
How does flow rate through a capillary depend on ΔP, D, L, and μ?
‹#›
MG Schrlau
Experimenting with Flow Through Pipe
Determine flow rate of fluids with different viscosities
through small capillaries having different diameters
and lengths
Time how long it takes you to suck out a given volume
through the capillaries
(1) Use a 0.9mm ID x 75mm long capillary to suck out
1.5ml of maple syrup (3X)  use this as the “standard”
(2) Repeat for 0.9mm ID x 100mm long and syrup
(3) Repeat for 0.7mm ID x 75mm long and syrup
(4) Repeat for 0.9mm ID x 75mm long and water
Calculate Experimental percentage for flow rate, D, L, and viscosity
 Parameternew  Parameterstandard 
Parameternew %  
 x100
Parameter
standard


‹#›
 0.7  0.9 
IDnew %  
 x100  22.2%
 0.9 
MG Schrlau
Pushing Fluids Through Channels
Simplified Bernoulli’s equation doesn’t capture all the losses, such
as friction of the fluid moving through the tube caused by viscosity.
Hagen-Poiseuille equation for flow rate through a tube (Re<<1)
Flow Rate (Q) Through a Tube:
 D 4 P
Q
128m L
P  pressure drop ( N / m 2 )
P1
D
Flow
P2
L  length (m)
D  tube inner diamter (m)
m  vis cos ity (kg / m  s )
L
P1 > P2
‹#›
MG Schrlau
Analyzing Our Results
Calculate Experimental percentage for flow rate, D, L, and viscosity:
Flowrateexpected
  D%  1 4 
%  
 1 x100
  100 



  100  
Flowrateexpected %   
  1 x100
  L%+100  
Flowrateexpected
  100  
%  
  1 x100
m
%+100
 

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MG Schrlau
Poiseuille Experiment
A simple controlled experiment is described by:
• M Dolz et al, European Journal of Physics 27 (2006), A laboratory
experiment on inferring Poiseuille's law for undergraduate students.
‹#›
MG Schrlau
Flow Through Very Nanochannels
Holt et al,
Science (2006)
1000X higher
flow rate than
continuum
predictions
Consistent with
molecular
dynamics
simulations
‹#›
MG Schrlau
Pushing Fluids Through Channels
Injecting fluids into cells requires a
finite volume of fluid be delivered.
Sophisticated microinjection
systems can pulse the applied
pressure for a given time, t.
Injection Volume Through a Tube (V):
 r 4 P
V
t
8m L
P  pressure drop ( N / m 2 )
L  length (m)
r  tube radius (m)
m  vis cos ity (kg / m  s )
MG Schrlau, Unpublished (2008)
• Too much volume will burst the cell
• Rule of Thumb: 1-1.5% of cell volume
 ~45 fl for a 20 μm diameter cell
t  time ( s )
‹#›
MG Schrlau
Pushing Fluids Through Cylindrical Channels
P1
P2
D
Flow
V
 r P
t
8m L
4
Assume :
L  20 m m
m  1 cP
L
P1 > P2
P=6000 hPa
D=400nm
1.2 s
5s
t
t
.4 s
1s
‹#›
MG Schrlau
Tools for Nanosurgery: Carbon-Based Nanoprobes
Bundled Nanotube Probe
Magnetically-Assembled Nanopipe Probe
Freedman et al (APL, 2007)
Nanotube-Tipped AFM Probe
Kouklin et al (APL, 2005)
Chen et al (PNAS, 2007)
• Requires specialized systems
• No connection to nanochannels
• Difficult, one-by-one assembly



Reinvestment in costly equipment
Unable to deliver fluids
Low yield, time consuming fabrication
‹#›
MG Schrlau
Carbon Nanopipettes (CNPs): An Integrated Approach
Integrates carbon nanopipes
into glass micropipettes
without assembly.
Carbon Tip
5 μm
Quartz Micropipette
Provides a continuous hollow,
conductive channel from the
microscale to the nanoscale.
Electrical
Connection
Quartz Exterior
Fits standard cell physiology
systems and equipment.
Inner
Carbon Film
Exposed
Carbon Tip
1 cm
Fabrication is amenable to
mass production for
commercialization.
Schrlau MG, Falls EM, Ziober BL, Bau HH, Nanotechnology, 2008
‹#›
MG Schrlau
The Integrated Fabrication of CNPs
(1)
(2)
(3)
Quartz
Pull catalyst-laden quartz
micropipettes
Catalyst
Deposit carbon inside by chemical
vapor deposition (CVD)
Quartz
Carbon
Wet etch the glass with BHF to
expose the carbon tip
2 Stages each loaded
with 100 CNPs
1 cm
Quartz
Carbon
Integrated Fabrication:
• Control tip outer diameter  Glass profiles
• 200 to 600 nm
• Control wall thickness  CVD time
• 30 to 80 nm for 2 to 4 hrs
• Control carbon length  Wet etching time / temp
• Eliminates assembly
• Hundreds produced in a single run, 98% efficiency
Schrlau MG, Falls EM, Ziober BL, Bau HH, Nanotechnology, 2008
‹#›
MG Schrlau
Estimating Injection Volume Through CNPs
3PL  1
V

8m  Geff

 t

 Lm  3 3  L  Lm  3 3
Geff  
  rm  ro   
  rL  rm 
 rm  ro 
 rL  rm 
RL = 200 nm
‹#›
MG Schrlau
CNP Properties and Capabilities as Cell Probes
Properties of CNPs
• Carbon structure – amorphous / graphitic
• Conductive from tip to tail  ~15 KΩ
• Transparent to light, electrons, and x-rays
• Elastic bending yet rigid for cell probing
Probing Cells with CNPs
• Cells remain viable when probed
• Cells continue to grow after being probed
• CNPs can effectively inject fluids into cells
10 μm
10 μm
Neurons 1 wk after injection
Schrlau MG, Falls EM, Ziober BL, Bau HH, Nanotechnology, 2008
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MG Schrlau
Additional Reading & References
Experiments
• M Dolz et al, European Journal of Physics 27 (2006), A laboratory
experiment on inferring Poiseuille's law for undergraduate students.
Good Review of All Things Nanotubes and Nanofibers
• Nanotubes and Nanofibers, Y Gogotsi (Ed.), CRC Press, 2006
Manuscripts
• Iijima, Nature, 1991 – discovery on carbon nanotubes
• Holt et al, Science, 2006 – flow through nanochannels
• Schrlau et al, Nanotechnology, 2008a – carbon nanopipettes
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MG Schrlau
Review of Today’s Topics
•
An overview of cells, intracellular components, and their functions
•
G10: Biology: Unit 3: Cell Structure and Function
•
•
•
•
Delivering material into cells – microinjection
•
G9: Phys Sci: Unit 6: Forces & Fluids
•
•
Cell Theory
Techniques of microscope use
Cell organelles – membrane, ER, lysosomes
Fluid pressure
Fluid transport through nanoscale channels
•
G9: Phys Sci: Unit 6: Forces & Fluids
•
•
Fluid pressure
G9: Phys Sci: Unit 11: Matter
•
Classifying matter
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MG Schrlau
Preview of Tomorrow’s Topics
•
Visualizing material transport and cellular response
• Light and optical microscopes
•
G10: Biology: Unit 3: Cell Structure and Function
•
•
Techniques of microscope use
G9: Phys Sci: Unit 10: Waves
•
•
Electromagnetic waves
Optics
• Molecules and fluorescence
•
G10: Biology: Unit 2: Introduction to Chemistry
•
•
G10: Biology: Unit 3: Cell Structure and Function
•
•
Chemistry of water
Techniques of microscope use
G9: Phys Sci: Unit 12: Atoms and the Periodic Table
•
•
•
•
Historical development of the atom
Modern atomic theory
Mendeleyev’s periodic table
Modern periodic table
• An example using Carbon Nanopipettes (CNPs)
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MG Schrlau