Micro/Bio-Engineering Universal Flu Vaccinesdosequis.colorado.edu/Courses/MicroNano/Lecture/Apr15.pdfBio/Nano/Micro-Engineering Universal Flu Vaccines Ryan Lewis, Ph.D. Dept of Mechanical

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

3

Flu Virus

4

Universal Vaccine

• M2 Protein: conserved between types

• Membrane-protein polyhedral Nano-particle

5

Bio/Micro Engineered Universal Flu Vaccine

6

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

7

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

8

Bio/Micro Engineered Universal Flu Vaccine

Reaction Zone

9

Micro Fluid Reaction--Detergent

Water Detergent

Oil

CMC

1. 2. 3.

1. 2. 3.

10

Micro Fluid Reaction--Lipid

Water Lipid

Oil

1. 2.

1.

3.

2. 3.

11

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

12

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

17

(≈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

18

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

19

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

21

Bio/Micro Engineered Universal Flu Vaccine

22

Removing Monomers

• Small particles diffuse faster

23

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.

32

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