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Bio/Nano/Micro-Engineering Universal Flu Vaccines
Ryan Lewis, Ph.D. Dept of Mechanical Engineering
University of Colorado at Boulder rjlewis@colorado.edu
Built on work by Ray Wu, Ph.D hsin-jui.wu@colorado.edu
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
3
Flu Virus
4
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
21
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
14
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
16
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?
20
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
24
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
25
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
26
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)
27
Device Microfabrication
28
Device Microfabrication • Step 1: photolithography
Fabrication processes of Photolithography
29
Device Microfabrication • Step 2: PDMS Soft Lithography
Fabrication processes of Softlithography
a. Oxygen plasma treatment on Glass slide
b. Oxygen plasma treatment on PDMS
30
Future work
Improvement of microfluidic devices Expend more inputs to test multi-condition.
Nanoparticle = ƒ(protein to lipid ratio, ph, salt concentration, buffer solution…)
31
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
33
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