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Bio/Nano/Micro-Engineering Universal Flu Vaccines
Ryan Lewis, Ph.D. Dept of Mechanical Engineering
University of Colorado at Boulder [email protected]
Built on work by Ray Wu, Ph.D [email protected]
1
Flu Virus
HA (hemagglutin)
NA (neuraminidase)
M1 (matrix protein #1)
M2 (matrix protein #2)
NP (nucleocapsid protein) and RNA
Lipid Bi-layer
PB1, PB2, PA (RNA polymerase)
NEP
2
Flu Vaccine
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Flu Virus
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Universal Vaccine
• M2 Protein: conserved between types
• Membrane-protein polyhedral Nano-particle
5
Bio/Micro Engineered Universal Flu Vaccine
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Manufacturing M2
M2 DNA Cloned DNA Transformed E. Coli
PCR
E.Coli with M2/MBP
M2/MBP Fraction M2+MBP
Cleave
Lots of M2, in a detergent micelle
Lab Chip. 2010 Oct 7;10(19):2519-26
PCR M2
Linking Proteins (MBP)
Transformation
M2
MBP
Amylose
incubate
http://biology200.gsu.edu/core_facility/InstrumentationRoomsNSC_Kell.html
Lyse, purify
MBP
M2
TEV
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M2 Virus-Like Particles
M. Vink et al. Journal of Structural Biology 160, 2007,295–304
Low concentration
High concentration
Diffusion
Protein/detergent
and lipid
Current method To get protein nano-particles, need: •Right protein: Lipid ratio •Right pH •Right buffer concentration •Right buffer solution •“Right dandruff”
With all correct conditions
•Week-long reaction time •(mm diffusion length)
•μL or mL sample, per test
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Bio/Micro Engineered Universal Flu Vaccine
Reaction Zone
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Micro Fluid Reaction--Detergent
Water Detergent
Oil
CMC
1. 2. 3.
1. 2. 3.
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Micro Fluid Reaction--Lipid
Water Lipid
Oil
1. 2.
1.
3.
2. 3.
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Micro Fluid Reactor Design • Flow-Rate Ratio
c∞=c0/(FRR)
5:1
win = wtot
6
20:1
win = wtot
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c=c0/5
c=c0/20
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Micro Fluid Reactor Design • Convective Diffusion
Total mass influx from diffusion
Total mass influx from convection
=
Δx
Δy
+
Mass gain on top
Mass loss on bottom
-
-
Mass gain in left
Mass loss out right
v
=0 13
Micro Fluid Reactor Design
• Convective Diffusion 0
– More accurate:
Need numeric solutions
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50 100 150 200 250 300 350 400 450
20
40
60
80
100020
4060
80100
0 0.5 1 1.5 2 2.5
Micro Fluid Reactor Design
• Convective Diffusion 0 • Assumptions:
– Very “spiky” initial concentration
– Infinite room to diffuse into
• Solution:
020
4060
80100
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
020
40
60
80
100
0
0.0
2
0.0
4
0.0
6
0.0
8
0.1
15
50 100 150 200 250 300 350 400 450
20
40
60
80
100
Micro Fluid Reactor Design
• Convective Diffusion 0 • Assumptions:
– Very “spiky” initial concentration
– Infinite room to diffuse into
• Solution:
As x-> ∞, c=c0win/wtot
020
40
60
80
100
0
0.0
2
0.0
4
0.0
6
0.0
8
0.10
2040
6080
1000 0.5 1 1.5 2 2.5 0
2040
6080
1000
0.05
0.1
0.15
0.2
0.25
0.3
0.35
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Calculation Summary
Valid everywhere Need simulation software
Good for wtot <
Good for wtot <
c=c0win/wtot=c0/FRR Good for
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(≈500 wtot for micelles)
Micro Fluid Reactor Design
• What it means to be successful. – FRR tuned to force detergent monomers and lipid micelles at x->∞ – Length long enough to allow reaction
Lipid(0.78mM)
Reaction zone
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Sample Calculation
• What is the max FRR? What is the min FRR?
• How far down the line does the reaction zone start? (using and a mid-point FRR)
Lipid Detergent
Initial Concentration 0.78 mM 2.5 mM
CMC 0.006 mM 0.17 mM
Diffusion coefficient 3x10-8 cm2/s 7.5x10-9 cm2/s
velocity 0.02 cm/s
Total width 0.01 cm
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Workshop
• Do the same calculations for a detergent whose CMC is 0.05 mM.
• How does increased velocity change things?
• What would happen if the detergent had a much larger diffusion coefficient?
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FRR 20 FRR 40
FRR 100
Experimental Results
MPP only observed
FRR 30 < MPP < FRR 130
No MPP –
When FRR < 30,
Detergent FC-14 above CMC.
When FRR > 130,
Lipid PC-14 below CMC.
FRR 120
FRR 60
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Bio/Micro Engineered Universal Flu Vaccine
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Removing Monomers
• Small particles diffuse faster
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Lookang Author of computer model: Francisco Esquembre, Fu-Kwun and lookang
Einstein-Stokes Relation
This is a simulation of Brownian motion of a big particle (dust particle) that collides with a large set of smaller particles (molecules of a gas) which move with different velocities in different random directions. http://weelookang.blogspot.com/2010/06/ejs-open-source-brownian-motion-gas.html
Vertical distance traveled by random walk:
Removing Monomers
• Detergent monomers diffuse to walls much faster
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50 100 150 200 250 300 350 400 450
20
40
60
80
100 0
0.05
0.1
0.15
50 100 150 200 250 300 350 400 450
20
40
60
80
1000.05
0.1
0.150.2
0.25
0.5 100.5 200.5 300.5 400.5 500.5
0.5
100.5
-5 -4 -3 -2 -1 0 1 2 3 4 5
x 10-3
0
0.005
0.01
0.015
0.02
0.025
Lipid concentration
Detergent
Micelles monomers
-5 -4 -3 -2 -1 0 1 2 3 4 5
x 10-3
0
0.005
0.01
0.015
0.02
0.025
Removing Monomers
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t=y2/D x=vt y2v/Dmon > x > y2v/Dmicelle
1 cm > x > 10 cm
1cm (rxn) 1-10cm (separation)
~3.5 cm
Protein-Lipid Ratios
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Nanoparticle = ƒ(protein to lipid ratio, ph, salt concentration, buffer solution…)
MscL : PC-14= 3:1 (30:1)
(1.575/0.05 mg/ml)
Experiment result -- We got MscL nanoparticles
MscL : PC-14 = 29:1 (290:1) (2.03/0.006 mg/ml)
MscL : PC-14 = 59:1 (590:1)
(2.0625mg/0.003mg)
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Device Microfabrication
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Device Microfabrication • Step 1: photolithography
Fabrication processes of Photolithography
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Device Microfabrication • Step 2: PDMS Soft Lithography
Fabrication processes of Softlithography
a. Oxygen plasma treatment on Glass slide
b. Oxygen plasma treatment on PDMS
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Future work
Improvement of microfluidic devices Expend more inputs to test multi-condition.
Nanoparticle = ƒ(protein to lipid ratio, ph, salt concentration, buffer solution…)
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After Micro-Engineering
• Test M2 Membrane-Protein Nano-particle as antigen in mice.
• Use microfluidics to zero-in on conditions to generate antigen MPN’s.
• Manufacture MPN’s using dialysis in industrial-scale drums.
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http://www.isumagazine.com/2011/09/to-conduct-animal-research-or-not/
Conclusions
• Nanoparticles for a universal flu vaccine
• Diffusion-based microreactors for nanoparticles
• Physics of convection/diffusion for micro-reactor design
• Microfabrication based on reactor design
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