1
Microfluidics – „Lab on a Chip“
Bi7430 Molecular Biotechnology
Outline
 introduction to microfluidics
 physics of micro-scale
 lab on a chip applications
 life and medical science
 protein and metabolic engineering
 design and fabrication
 sensing and detection
Lab on a Chip Concept
p r e p a r a t i o n
a n a l y s i si n c u b a t i o n
c o l l e c t i o n
p r e - t r e a t m e n t
Microfluidics
 „ b e h av i o r, c o n t ro l a n d m a n i p u l a t i o n o f f l u i d s g e o m e t r i c a l l y
c o n s t ra i n e d t o a s m a l l d i m e n s i o n s “
 d i m e n s i o n s ( 1 ´ - 1 0 0 ´ m m )
 v o l u m e s ( n L , p L , f L )
 u n r i v a l l e d p r e c i s i o n o f c o n t r o l
 ( u l t r a ) h i g h a n a l y t i c a l t h r o u g h p u t
 r e d u c e d s a m p l e a n d p o w e r c o n s u m p t i o n
 f a c i l e p r o c e s s i n t e g r a t i o n a n d a u t o m a t i o n
Nature 507, 181 (2014)
2
Revolution in Electronics
Size (nm) Price (USD)
Vacuum tube 100 10
Trasistors 10 1
Microchip 0.000 010 0.000 000 100
Revolution in Science?
Volume (mL) Throughput (assays/day)
Test tube 1 000 10
Microtiter plate 100 1 000
Microfluidic chip 0.000 001 1 000 000
Nature Biotechnol. 21, 1179 (2003)
 c o n t i n u o u s - f l o w m i c ro f l u i d i c s
m a n i p u l a t i o n o f c o n t i n u o u s l i q u i d f l o w
t h r o u g h m i c r o - f a b r i c a t e d c h a n n e l s
 d ro p l e t - b a s e d m i c ro f l u i d i c s
m a n i p u l a t i n g d i s c r e t e v o l u m e s o f f l u i d s
i n i m m i s c i b l e p h a s e s
 d i g i t a l m i c ro f l u i d i c s
d r o p l e t s m a n i p u l a t e d o n a s u b s t r a t e
u s i n g e l e c t r o - w e t t i n g
Concepts in microfluidics Novel Physics of Micro-Scale
 v i s c o s i t y, s u r f a c e t e n s i o n a n d c a p i l l ar y f o rc e s d o m i n a t e
 l a c k o f t u r b u l e n t p h e n o m e n a
+ n o n t r i v i a l c h e m i c a l g r a d i e n t s
t o s t u d y c h e m o t a x i s
 a b s e n c e o f d e n s i t y - d r i v e n c o n v e c t i o n
+ f r e e i n t e r f a c e d i f f u s i o n , e f f i c i e n t
p r o t e i n c r y s t a l l i z a t i o n k i n e t i c s
 s t r o n g s h e a r i n g f o r c e s
+ f a s t m i x i n g k i n e t i c s o f p r o t e i n
f o l d i n g a n d / o r c a t a l y s i s
Nature Biotechnol. 20, 826 (2002) PNAS 99, 16531 (2002)Appl. Phys. Lett. 83, 4664 (2003)
3
Lab on a Chip applications
 a n a l y t i c s a n d c h e m i s t r y
 P C R a n d s e q u e n c i n g
 p o i n t o f c a re d i a g n o s t i c s
 p h a r m a c o l o g y
 c l i n i c a l s t u d i e s
 s i n g l e c e l l b i o l o g y
 h i g h t h ro u g h p u t b i o l o g y
Polymerase chain reaction
 c l a s s i c a l P C R
 s l o w h e a t i n g / c o o l i n g c y c l e s
 P C R t u b e s ( s t r i p s ) , 9 6 - w e l l M T P
 v o l u m e 5 0 t o 5 0 0 m L
K a r y M u l l i s
N o b e l P r i z e i n 1 9 9 3
Digital polymerase chain reaction
 d i g i t a l P C R
 1 n a n o l i t e r d r o p l e t s
 2 0 0 0 0 d r o p l e t s p e r r u n
Next-generation sequencing
 p a ra l l e l i za t i o n o f s i n g l e m o l e c u l e p y ro s e q u e n c i n g
 4 5 4 P y ro s e q u e n c in g ( R o c h e )
w a t e r i n o i l d r o p l e t s 1 p i c o l i t e r ( 1 0 - 1 2 l i t e r s )
1 m i l . r e a d s / r u n , 1 0 U S D / M b a s e
F r e d e r i c k S a n g e r
N o b e l P r i z e i n 1 9 8 0
4
 2 0 0 3 : 1 3 y e a r s , 3 b i l l i o n U S D
 2 0 1 6 : d a y s , < 1 , 0 0 0 U S D
H G P
R o c h e
4 5 4
I l l u m i n a
H i S e q X
Revolution in Science? Organs on chip
 3 D c h i p s m i m i c k i n g h u m a n ’s p h y s i o l o g ic a l re s p o n s e s
( e . g . , p a t h o l o g i c al , p h a r m a c o k i n e t i c , t ox i c o l o g i c a l )
 re a l i s t i c i n v i t r o m o d e l c l o s e r t o i n v i v o c e l l e n v i ro n m e n t
( e . g . , m e c h a n i c a l s t ra i n , p a tt e r n i n g , f l u i d s h e a r s t re s s e s )
 c a n re p l a c e e x p e n s i v e a n d c o n t ro v e rs i a l a n i m a l t e st i n g
Nature 471, 661–665 (2011) Biophysical Journal 94(5) 1854–1866
m i c ro p i l l a rf l a t s u r f a c e
L u n g
H e a r t
S p l e e n
B o n e
N e u r o v a s c u l a r
A r t e r y
K i d n e y
I n t e s t i n e
Organs on chip Human on chip concept
 c o r r e c t l i m i ta t i o n s o f o r g a n s i s o l a t i o n
 w h o l e b o d y b i o m i m e t i c d e v i c e s
5
Sequence diversity
No. Coverage (95%)
1 94
2 3 066
3 98 163
4 3 141 251
5 100 520 093
Unexplored
proteindiversity
(Ultra)High-throughput screening
Lab Chip 2009, 9: 1850 Anal. Chem. 2014, 86: 2526
Robotic mFluidic
Reaction volume 100 mL 5 pL
Reactions / day 50 000 1 . 108
Total time 5 years 3 days
Total volume 5 000 L 150 mL
No. of plates / devices 250 000 2.0
No. of tips 28 000 000 10
Enzyme specificity profiling
m Fluidics
Robotics
Conventional
Conventional Robotic mFluidic
Reaction volume (mL) 10 1 0.00015
Total enzyme (mg) 540 54 0.5
Total time (days) 100 30 5
Anal. Chem. 2013, 85: 4761 Michal Vasina, CeCe Junior, Wednesday 10:15
Steady-state kinetics
Concentration(mM)
Tempertaure(°C)
m Fluidic
Conventional
Conventional mFluidic
Reaction volume (mL) 2 0.00010
Total enzyme (mg) 1 0.01
Throughput per hour 5 10 000
Small 2015, 11: 4009
6
Mechanism and thermodynamics
Mixing
Time (ms)
Signal
Time (s)
E + S ES EI EP E + P
Energy
Stopped-flow mFluidic
Dead time 0.3 ms 0.7 ms
Reaction volume 100 mL 10 pL
Temp. equilibration 10 min 50 ms
Signal integration 0.5 ms no limit
Multienzyme Systems
H L D H H D E H H H D E H
ChemBioChem 15: 1891 (2014) ACS Synth. Biol. 3: 172 (2014)
E s c h e r i c h i a
R h o d o c o c c u s A g r o b a c t e r i u m
Multienzyme systems
 1 n L d r o p l e t v o l u m e
 1 0 0 0 0 a s s a y s / h o u r
H L D H H D E H H H D E H
r o b o t
s c i e n t i s t
Design and fabrication
 soft lithography originates from semiconductor industry
DESIGN / MODELING MASK / MOLD CASTING / BONDING
7
Design and fabrication
 direct fabrication methods
3D PRINTING LASER CUTTING CNC m-MILLING
Design and fabrication
 m a t e r i a l s
 i n e r t a n d t r a n s p a r e n t
 P D M S - p o l y ( d i m e t h y l s i l o x a n e )
 P M M A - p o l y ( m e t h y l m e t h a c r y l a t e )
 f u s e d s i l i c a , q u a r t z a n d g l a s s
 s u r f a c e m o d i f i c a t io n
 p l a s m a t r e a t m e n t
 s i l a n i z a t i o n
 s o l – g e l c o a t i n g
Sensing and detection
 p ro c e s s i n g o f s m a l l re a g e n t v o l u m e s
 a n a l y t i ca l t i m e s c a l e a n d p e r f o r m a n c e
 o n c h i p d e t e c t i o n
 f l u o r e s c e n c e ( L S M , F C S , F L I M )
 U V/ V I S a b s o r b a n c e
 I R s p e c t r o s c o p y
 R a m a n s c a t t e r i n g
 ( c h e m o / e l e c t r o ) l u m i n e s c e n c e
 t h e r m a l c o n d u c t i v i t y
 R I v a r i a t i o n
 o f f c h i p d e t e c t i o n
 G C , H P L C , M S
 N M R , X - r a y
Commercial Solutions
 c u s t o m i z e d d e s i g n a n d f a b r i c a t i o n
 e n t i r e t e c h n o l o g i e s
Nature 499, 505 (2013)Nature Meth. 10, 1003 (2013)
8
Conclusions
 r e d u c e d s a m p l e / r e a g e n t / p o w e r c o n s u m p t i o n
 s u p e r i o r p e r f o r m a n ce a n d n o v e l p h y s i c s
 a p p l i c a t i o n s i n l i f e a n d m e d i c a l s c i e n c e s
 i n - h o u s e a s w e l l a s c o m m e r c i a l t e c h n o l o g i e s
m ic r oflu id ic s r ev olu t ion iz e s c ien c e
Reading
 Yu m , K . , 2 0 1 4 : P h y s i o l o g i c a l l y re l e v a n t o rg a n s o n c h i p s .
B i o t e c h n o l . J . 2 0 1 4 , 9 , 1 6 – 2 7
 2 . K e y e l e m e n t s o f m i c r o e n v i r o n m e n t s ( p a g e 1 8 - 2 2 )