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
▪ discovery of novel proteins
▪ 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 a v 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)
Miniaturization & integration
vacuum tubes transistors micro chips
size (mm) 100 10 0.000 01
price (USD) 10 1 0.000 000 1
test tube microtiter plate μ-fluidic chip
volume (μL) 1 000 10 0.000 001
throughput (assays/day) 10 1 000 1 000 000 ?
❑ c o n t i n u o u s - f l o w m i c r o 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 r o p l e t - b a s e d m i c r o 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 fa c e t e n s i o n a n d c a p i l l a r y fo 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)
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 ( s i n g l e m o l e c u l e )
▪ 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
▪ f a s t h e a t t r a n s f e r
Analytical Chemistry, 2008, 80, 8975
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 r o s e q u e n c i n g ( Ro 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
❑ 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 8 : 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
Revolution in DNA analysis
Organ(oid)s 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 hy s i o l o g i c a l re s p o n s e s
( e . g . , p a t h o l o g i c a l , 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 r o n m e n t
( e . g . , m e c h a n i c a l s t ra i n , p a t t e r n i n g , f l u i d s h e a r s t re s s e s )
Nature 471, 661–665 (2011) Biophysical Journal 94(5) 1854–1866
m i c r o p i l l a rf l a t s u r fa 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
Organ(oid)s 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 hy s i o l o g i c a l re s p o n s e s
( e . g . , p a t h o l o g i c a l , 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 r o n m e n t
( e . g . , m e c h a n i c a l s t ra i n , p a t t 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 r e 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 s t i n g
Nature 471, 661–665 (2011) Science 364, 960-965 (2019)
UNEXPLORED
DIVERSITY
Nucleic Acids Res. 48, W104–W109 (2020)
Protein Discovery and Engineering
F U N C T I O N A L C H A R A C T E R I S AT I O N
No. Coverage (95%)
1 94
2 3 066
3 98 163
4 3 141 251
5 100 520 093
Protein Discovery and Engineering
S C R E E N I N G A N D I D E N T I F I C AT I O N
O F P O S I T I V E H I T S
F U N C T I O N A L C H A R A C T E R I S AT I O N
(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
Conventional
μFluidicsConventional μFluidics
Activity and specificity in μ-droplets
μFluidics
Robotics
Conventional
Anal. Chem. 2019, 91: 10008-10015
Test tube Robotic μFluidic
Reaction volume (mL) 10 1 0.000 15
Total enzyme (mg) 540 54 0.5
Total time (days) 100 30 5
ACS Catalysis (in review)
IN SILICO SCREENING HIGH-THROUGHPUT CHARACTERISATION
Activity and specificity in μ-droplets
Concentration
(mM)
Tempertaure(°C)
Small, 11: 4009 (2015)
Steady-state kinetics in μ-droplets
Test tube μFluidic
Reaction volume (mL) 10 0.000 1
Total enzyme (mg) 1 0.01
Throughput per hour 50 10 000
μFluidic
Conventional
ChemBioChem 15: 1891 (2014) ACS Synth. Biol. 3: 172 (2014)
R h o d o c o c c u s A g r o b a c t e r i u m
Synthetic biology
Small, 11: 4009 (2015)
Enzyme cascades in μ-droplets
AI in protein engineering
ITERATIVE OPTIMIZATIO N
Bayesian optimization
MOLECULAR DYNAMI CS
unsuper vised learning
Nat. Comm. 9 (2018)
STRUCTURE PREDIC TION
deep learning
deepmind.com
SEQUENCE BASED PREDIC TION
super vised learning
STRUCTURAL BIOLOGY
Multidisciplinary science
MICROFLUIDICS
COMPUTATIONAL DESIGN
ENZYME KINETICS ARTIFICIAL INTELLIGENCE
Design and fabrication
❑ soft lithography originates from semiconductor industry
DESIGN / MODELING MASK / MOLD CASTING / BONDING
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 fa c e m o d i f i c a t i o 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 r o c e s s i n g o f s m a l l r e a g e n t v o l u m e s
❑ a n a l y t i c a l t i m e s c a l e a n d p e r fo 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 instruments and services
❑ 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)
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 c e 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 i c r o f l u i d i c s r e v o l u t i o n i z e s c i e n c e & t e c h n o l o g y
Reading
❑ Yu m , K . , 2 0 1 4 : P hy s i o l o g i c a l l y r e l e v a n t o r ga n s o n c h i p s .
B i o te c h n o l . J . 2 0 1 4 , 9 , 1 6 – 2 7
❑ 2 . Ke y e l e m e n t s o f m i c r o e nv i r o n m e n t s ( p a g e 1 8 - 2 2 )