• Cells
• Microscopes
• Light
• Atoms
• Cell structure
• Electromagnetic radiation
• Atomic models
• Fluid transport
• Messenger-mediated calcium signaling
• Cell microinjection
• Carbon nanotubes
• Binding energy
‹#›
• Carbon nanopipettes
• IP3, NAADP, cADPr
• Hagen-Poiseuille
• Stokes’ shift
MG Schrlau

Michael G. Schrlau
Mechanical Engineering and
Applied Mechanics
University of Pennsylvania

About Me - Background
University of Pittsburgh
(BSME, 1998)
Elizabeth Jean
‹#› 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)
‹#› 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MG Schrlau, Unpublished (2008)
‹#› 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
MG Schrlau et al, Nanotechnology (2008a)
HRTEM
Intracellular Calcium Signaling
Targeting Before Injection After Injection
MG Schrlau et al, Nanotechnology (2008b)
‹#› 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

Fitting the Topics into the High School Curriculum
• An overview of cells, intracellular components, and their functions
• G10: Biology: Unit 3: Cell Structure and Function
•
Cell Theory
•
Techniques of microscope use
• Cell organelles – membrane, ER, lysosomes
• Delivering material into cells – microinjection
• G9: Phys Sci: Unit 6: Forces & Fluids
•
Fluid pressure
• Fluid transport through nanoscale channels
• G9: Phys Sci: Unit 6: Forces & Fluids
• Fluid pressure
• G9: Phys Sci: Unit 11: Matter
• Classifying matter
‹#› MG Schrlau

Fitting the Topics into the High School Curriculum
• 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
•
Chemistry of water
• G10: Biology: Unit 3: Cell Structure and Function
• 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)
‹#› MG Schrlau

‹#› MG Schrlau

‹#›
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
‹#› 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 White & red blood cells
Neuron cell Bone cells http://epigenome.eu/media/images/large/34.jpg
bp0.blogger.com
‹#› www.immediart.com/catalog/images/ big_images/SPL_ItB_P360276-Granule
_nerve_cell,_SEM.jpg
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
‹#› 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
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
‹#› MG Schrlau

www. jupiterimages.com
Areas in Macroscale Surgery
Cutting
Material
Delivery
Macroscale Surgery www.south-norfolk.gov.uk
www. jupiterimages.com
Manipulating
‹#›
Sensing www.altramedical.com
MG Schrlau

Sizing-Down Surgery
Investigate a problem or repair-replace-modify a component
3m 60 μm
100 μm
Cells
10 μm
Nucleus, Organelles
1 μm
Proteins, Cytoskeleton, DNA
100 nm 10 nm 1 nm
Surgery can be performed on cells using tools with nanoscale resolution
‹#› MG Schrlau

Shen et al (MCB, 2005)
Developing Areas of Cell Nanosurgery
Cutting
Material
Delivery
Schrlau (Unpublished)
Cell Nanosurgery
Kim and Lieber (Science, 1999)
Manipulating
‹#›
Sensing
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
• Fluid Delivery
• Through nanochannels
• Minimally invasive to cells
• Minimal damage to cells
MG Schrlau, 2008, unpublished
‹#› www.gamasutraexchange.com/FullPreview/Index.cfm/ID/2141
04/intType/7/stgCHSource/Popular
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 DNA-
GFP Plasmid
‹#› 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.
Deliver molecule
Molecule binds to some organelle
MG Schrlau, 2008, unpublished
Organelle releases calcium
?
?
‹#› MG Schrlau

Delivering Microscopic Material to Cells
Mouse embryos (4 day blastocyst) injected with embryonic stem (ES) cells
ES Cells (~15 μm diameter) can be easily resolved with visible light
(400-700nm)
‹#› 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
‹#› MG Schrlau

Injection-Mediated Intracellular Calcium Signaling
Inverted Microscope (Nikon)
Perfusion System
CCD Camera (Roper)
Manipulator
(Eppendorf)
Filter Wheel
(Sutter)
Injection System
(Eppendorf)
Ex Em
Breast cancer cells
(SKBR3) loaded with Fura-2AM
Ex: 340, 380 nm
Em: 540 nm
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

G10: Biology: Unit 3: Cell Structure and Function
‹#› 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
‹#› MG Schrlau

Anatomy of a Cell http://people.eku.edu/ritchisong/RITCHISO/301notes1.htm
= Membrane-bound organelles
‹#› MG Schrlau

Anatomy of a Cell
Molecular Biology of the Cell, 4 th Edition
‹#› MG Schrlau

Plasma Membrane
Lipids are amphiphilic
•
Likes water (hydrophilic)
• Dislikes water (hydrophobic)
Very flexible; large deflections
Permeability barriers - water-soluble solutes cannot pass freely across the lipid bilayer
Anchoring cells to surfaces
‹#› 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
‹#› MG Schrlau

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 www.itg.uiuc.edu/technology/atlas/structures/nucleus/
‹#› http://cellbio.utmb.edu/cellbio/nucleus.htm
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
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
‹#› 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
‹#› 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
‹#› 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
‹#› MG Schrlau

Why Study 2 nd Messengers & Calcium Signaling?
Unregulated calcium release implicated in cancer – only IP3 has been studied
(Monteith et al, Nat Rev Cancer, 2007)
Some Second Messengers:
• IP
3
– Inositol triphosphate
• cADPr – Cyclic adenosine diphosphate ribose http://people.eku.edu/ritchisong/RITCHISO/301notes1.htm
• NAADP – Nicotinic acid adenine dinucleotide phosphate
Calcium Stores:
• Endoplasmic Reticulum (ER) – sensitive to IP3 and cADPr (in some cells)
• Lysosomes (Ly) – sensitive to NAADP**
‹#› MG Schrlau

In Short, Cells are Complex!
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.
‹#› http://www.medifast1.com/shopping/images/products/stew_large.jpg
www.daylife.com/photo/0fcw2UV6LU9Hq
MG Schrlau

Additional Reading & References
• • www.cellsalive.com/cells/cell_model.htm
HyperLink
• Alberts, Molecular Biology of the Cell, 4 th Edition, Garland Science, 2002
‹#› MG Schrlau

G9: Phys Sci: Unit 6: Forces & Fluids
‹#› 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
 Cell is crowded space – organelles could be damaged or destroyed
Intracellular
150mM K +
20mM Na +
4mM Cl -
0.1
μM Ca ++
Extracellular
4mM K +
145mM Na +
110mM Cl -
2mM Ca ++
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
‹#› 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
1)
Introduce virus
Plate of cells
Cells
2) http://fig.cox.miami.edu/~cmallery/150/gene/sf11x1virus.jpg
4)
3)
‹#›
Virus
Infected cells
MG Schrlau

Viral Transduction (or Infection)
Advantages Very efficient, Can modify many cells (>thousands)
Disadvantages Safety concerns, can’t delivery drugs
‹#› 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
‹#› 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
‹#› MG Schrlau

Non-Viral Transfection
Ex: Magnetofection
Phagocytosis
• Solid material comes in contact with cell (gravity, centrifuge, magnet)
Cell brings in the solid particle 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
‹#› 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
‹#› MG Schrlau

Projectile Delivery
Gene Gun
Non-Viral Transfection 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
‹#› MG Schrlau

Non-Viral Transfection for Single Cells
Single-Cell Electroporation
Makes membrane permeable
(presumably new holes) to external molecules.
Olofsson et al, Curr Opin Biotechnol, 2003
• Cell populations or single cells
• No control of concentration, semi-elaborate transfection setup
‹#› 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
Laser
Larger Pipette with
DNA in fluid
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
‹#› 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
Cylindrical Nanochannel
EXTRACELLULAR
Fluid inside
Submicron Diameter
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
‹#› MG Schrlau

The Many Ways of Delivering Material into Cells
Viral transfection
• Very efficient
• Safety concerns
Liposomes
Non-Viral Transfection:
• Eliminates safety concerns
• Not as efficient
Charged
Copolymers
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
‹#› MG Schrlau

G9: Phys Sci: Unit 6: Forces & Fluids
G9: Phys Sci: Unit 11: Matter
‹#› MG Schrlau

Glass Micropipettes
Injection of Fluids
Submicron Diameter www.eppendorfna.com
Advantages
• Widely-used, most trusted
• Platform technology for modern cell physiology
•Relatively easy to make
•Low cost
Disadvantages
• Single function
• Fragile
• Large for nanosurgery
• Invasive
• Can cause irreparable damage to cell membrane
‹#› MG Schrlau

Microinjection Through Nanochannels
Concept of Microinjection
Cylindrical Nanochannel
EXTRACELLULAR
Fluid inside
Nanochannel connected to larger probe
INTRACELLULAR
Cell Membrane
Simplified Model of Microinjection
P injection
Flow
P
1
Flow
L
P
1
> P
2
D
‹#›
P
2
MG Schrlau

Carbon Nanotube and Nanopipes
Carbon Nanotubes Carbon Nanopipes
Iijima (Nature, 1991)
‹#›
Whitby and Quirke
(Nat. Nanotech, 2007)
Minimally invasive probes for material delivery and sensing
• High aspect ratio
• Nanoscopic channels
• High mechanical strength
• High electrical conductivity
MG Schrlau

Carbon Nanotubes and Carbon Nanopipes
Carbon Nanotubes
Carbon nanotubes (CNTs)
• First discovered by Iijima (1991)
Single Wall
(SWCNT)
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
‹#›
Iijima (Nature, 1991)
Multi Wall
(MWCNT) www.reading.ac.uk/Physics/ pgprogrammes/MSCprojects2008.htm
MG Schrlau

Carbon Nanotubes and Carbon Nanopipes
MWCNT
SWCNT - Diameters of 0.6 to 1.8 nm, lengths of 20 nm to 500 m m, high strength, electrical and thermal conductivity
MWCNT - Diameters >0.6 nm, lengths of 20 nm to 500+ m m, high strength, electrical and thermal conductivity
10 nm
Rossi et al, Nano Lett. (2004) www.nano-lab.com/nanotube-image3.html
Carbon Nanopipes
• Diameters >50nm, lengths of 20 nm to 500+ m m, high strength, electrical and thermal conductivity
• 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) www.borealisgroup.com/images/infrastructure/pipes-fittings/PE100_water_pipe.jpg
‹#›
Courtesy of E Vitol, Gogotsi Group, Drexel University, 2008
MG Schrlau

Pressure Injection Systems
Narishige Piston Injector Eppendorf Femtojet Injector www.narishige.co.jp/products/group1/im-9b.htm
• Piston displacement controls pressure and volume
‹#› www.eppendorfna.com
• Pressure is set and pulse time controls volume
• Capable of 6000 hPa
MG Schrlau

Pushing Fluids Through Channels
Reynolds Number (Re):
Re

m



( )

( / )
 m

3
( / )

cos ( /

)
Re>>1: Inertia dominates  Turbulence
Re=1: Equal contribution
Re<<1: Viscosity dominates  Laminar
**In micro/nano environments, typically Re<<1
‹#› MG Schrlau

Determining Flow Through a Pipe
2D Flow Profile
P
1
Flow (Q), P
1
> P
2
μ
L
D P
2
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
Parameter new
%

Parameter new

Parameter standard
Parameter standard

 x 100
‹#›
ID new
%

0.9
x 100
 
22.2%
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)
P
1
Flow Rate (Q) Through a Tube:
Flow
P
1
> P
2
L
D P
2
Q


D
4 
P
128 m
L
P pressure drop N m
2
( / )
L

( )
D

( ) m  vis cos ( /

)
‹#› MG Schrlau

Analyzing Our Results
Calculate Experimental percentage for flow rate, D, L, and viscosity:
Flowrate expected
%



D
100
4

1

 x 100
Flowrate expected
%


 
100
L%+100 

1

 x 100
Flowrate expected
%





100 m
%+100



1

 x 100
‹#› 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):
V


8 r 4 
P m
L t
P pressure drop N m 2 ( / )
L

( ) r

( ) m  vis cos ( /

) t

( )
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
‹#› MG Schrlau

P
1
Pushing Fluids Through Cylindrical Channels
Flow
P
1
> P
2
L
D=400nm
D P
2 V

 r
4 
P
8 m
L t
Assume :
L

20 m m

1 cP m
P=6000 hPa
5 s t
1 s
1.2 s t
.4 s
‹#› 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  Reinvestment in costly equipment
• No connection to nanochannels  Unable to deliver fluids
• Difficult, one-by-one assembly  Low yield, time consuming fabrication
‹#› MG Schrlau

Carbon Nanopipettes (CNPs): An Integrated Approach
Carbon Tip Integrates carbon nanopipes into glass micropipettes without assembly.
5 μm
Quartz Micropipette
Electrical
Connection
Provides a continuous hollow, conductive channel from the microscale to the nanoscale.
Quartz Exterior
Fits standard cell physiology systems and equipment.
Inner
Carbon Film
Exposed
Carbon Tip
1 cm
Schrlau MG, Falls EM, Ziober BL, Bau HH, Nanotechnology , 2008
‹#›
Fabrication is amenable to mass production for commercialization.
MG Schrlau

The Integrated Fabrication of CNPs
(1) Pull catalyst-laden quartz micropipettes
Quartz
Catalyst
Quartz
Carbon (2) Deposit carbon inside by chemical vapor deposition (CVD)
(3) 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
V

3
 
8 m
P
L


1


 G eff
 t
RL = 200 nm
G eff



L m r m
 r o


 r

3 m
 r o

3




L

L m r
L
 r m


 r
L

3  r m

3

‹#› MG Schrlau

CNP Properties and Capabilities as Cell Probes
Properties of CNPs
• Carbon structure – amorphous / graphitic
• Conductive from tip to tail  ~15 K Ω
• 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
‹#› 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
‹#› MG Schrlau

Review of Today’s Topics
• An overview of cells, intracellular components, and their functions
• G10: Biology: Unit 3: Cell Structure and Function
•
Cell Theory
•
Techniques of microscope use
• Cell organelles – membrane, ER, lysosomes
• Delivering material into cells – microinjection
• G9: Phys Sci: Unit 6: Forces & Fluids
•
Fluid pressure
• Fluid transport through nanoscale channels
• G9: Phys Sci: Unit 6: Forces & Fluids
• Fluid pressure
• G9: Phys Sci: Unit 11: Matter
• Classifying matter
‹#› 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
•
Chemistry of water
• G10: Biology: Unit 3: Cell Structure and Function
• 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)
‹#› MG Schrlau