A Lab-on-Chip System for direct SNP
sensing from human blood
Ichiro Yamashita1,3, Paolo Fiorini2
1Advanced
Technology Research Laboratory, Panasonic corp.
3-4 Hikaridai, Seika-cho, Soraku-gun, Kyoto 619-0237, Japan
2Imec, Kapeldleef 75, B-3001, Leuven, Belgium
3Nara Institute of Science and Technology, 8916-5 Takayama-cho Ikoma
1
2
What is SNP
One DNA alternation can affect people’s sensibility against drugs
A G T C C G G G C G
T C A G G C C C G C
A G T C C A G G C G
T C A G G T C C G C
ＳＮＰ
predisposition to diseases,
difference in reaction on drugs
(ie. adverse side effects), etc
Single Nucleotide
Polymorphism
3
SNP sensor by Panasonic
DNA
A
Electrode
Target DNA from
a patient
Patient
Target DNA
fixation
is not necessary
No SNP exist
SNP exists
replication
No replication
Current
phosphate
compound
enzymatic
reaction
e-
enzymatic
reaction
4
Easy and fast detection of SNP
Outsourcing
Service
Present：manual handling )
DNA
extraction
DNA amp.
（PCR）
DNA
purification
Large Equipments
detection
（several
business days）
SNP
judgment
Our target is SNP-detection from blood
less than 1 hour with small one-chip.
5
SNP Detection chip
DNA amp.
（PCR）
DNA
extraction
Pump
DNA
purification
detection
SNP: A
Microfluidic chip
Buffer
SNP: B
Pump
Valve
SNP: C
Mixing
SNP: D
DNA filter
PCR
Blood
1uL
SNP: E
Pump
SNP sensing
Rapid genotyping diagnostic on chip system
• Automated DNA extraction from blood
• Output diagnostic results within 1 hour
6
SNP Detection chip
Pump
SNP: A
Buffer
SNP: B
Pump
Valve
SNP: C
Mixing
DNA filter
SNP: D
PCR
Blood
1uL
1cm
Pump
SNP: E
SNP sensing
7
Single SNP Detection chip details
Blood
Reagent 2
Pump
Mixer 2
Mixer 1 PCR 1
PCR 2
Detector
Reagent 1
Valve
Module 1
Sample
loading
Drain
DNA extraction
and amplification
Valve
Coarse
filter
Module 2a
Allele specific
amplification
Drain
Electro‐
chemical detection
8
Single SNP Detection Chip
PCR
Pump
Valve
Detector
8
Single SNP Detection device
Mixer 1
PCR 1
Mixer 2
Blood
Coarse filter
Pump
Valve
PCR 2
Pump
Detector
Reagent 2
Mixer 2 PCR 2
Mixer 1 PCR 1
Reagent 1
Module 1
Valve
Valve
Coarse
filter
Drain
Detector
Module 2a
Drain
Single SNP Detection device
Mixer 1
PCR 1
Mixer 2
Coarse filter
Pump
PCR 2
Plastic layer
Reservoir
Valve
9
Pump
Valve
Detector
Silicon layer
Valve
Mixer
Thermal
solution
PCR
Pyrex layer
10
11
Fabrication SSD (Process flow)
Blanket Si wafers
1 µm therm oxide
Pyrex wafer
Anodic bonding
Grinding and cleaning
BS oxide removal wet
IX-litho
Litho
Oxide etch
Si etch, Strip
Etch and strip
11
Fabrication SSD (Results)
12
Coarse filter
FS looking through glass
PCR chamber
Coarse filter
~300µm
BS Si surface
Mixer
12
The newly developed pump unit
• Operation pressure 3MPa
13
・1.5V battery drive
• Maximum generation pressure 30MPa
+
+
electrode
Pumping
action
electrode
case
Layered polymer
actuator
Silicon sub.
Diaphragm
Conductive Polymer Actuator
14
Operating principle：
volume change by injection/
extraction of electrolyte ions
sewell
shrink
Polymer
Electrolyte
EMI-TFSI
V
－
Polymer:
－
Reduce
polypyrrole
(PPy) base
with TFSI
+
－
－
Oxid
+
+
PPy-TFSI (charge: neutral)
N
N
－
N
＋
－
－
N
N
reduction
Py+  Py0
N
－
－
－
EMI+
N
N
－
+
N
+
Battery
+ Electric power
EMI
+
－
Distortion
up to 10％ OK
negative charge attracts positive
ions from electrolyte.
N
D: diffusion constant
DEMI+ > DTFSI-
15
Polymer pump characterization
Adhesive dots
Strain (%)
3
inactive CP
dot Glue
active CP
metal
whole Glue
Load [MPa]
0.5
1.0
2.0
7.0
10.0
2
1
15.0
0
0
50
100
Time (s)
1cm
Mixers : characterization
Mixing of two fluids
inlet
Mixing of particles
(simulating blood cells)
inlet
Outlet
Port
Inlet
Port 1
outlet
Inlet
Port 2
16
PCR demonstration & characterization
PCR chamber part of the LoC
17
PCR chamber
Filled with sample
Thermal isolation is the key
Least heat current should be realized between
substrate and PCR chamber during PCR cycle
The chamber is isolated and connected only
with the tiny micro-channel
On chip PCR: fabrication and thermal tests
Thermal insulation is further improved by winding in/out
channels around the cavity.
Section View A-A
z
x
Cover Glass
Silicon Walls
Liquid
A
A Thermal
Isolation
Gap (Air)
Temperature Difference, T-Tamb (K)
80
70
PCR Temp.
60
Chip Temp.
Max 10 K
above RT
50
40
30
20
10
0
-10
-20
-1.2
-0.8
-0.4
0
0.4
0.8
1.2
TEC Current (A)
Steady‐state temperature of the PCR chamber and bulk silicon substrate.
18
19
Fast Micro-PCR Demonstration
100
95
Temperature (°C)
Template: Whole Blood
100
Temperature (°C)
90
80
70
60
90
85
80
75
MicroPCR
result
70
65
60
0
20
40
60
80 100 120
Time (s)
50
40
30
20
0
5
10
15
20
Time (min)
 Successful micro-PCR amplification in
less than 21 minutes -> 11 minutes.
SSD Amplification Tests (Human Genomic DNA)
Template: Human Genomic DNA
(From Commercial Source)
Protocol
2X buffer KOD fx
2 mM dNTP’s 10 M Fw primer
10 M Rv primer
KOD polymerase
Water
Template
Total
5 L
1 L
1 L
1 L
0.4 L
1.1 L
0.5 L
10 L
(2 L amplified
in micro‐PCR)
MicroPCR
Negative
Control
Positive
Control
DNA
Ladder
T anneal = 60°C
# Cycles = 30
Commercial Tool SSD
 Successful
amplification of
Test chip is preloaded with PCR buffer target fragment in micro-PCR
solution
 Reproducibility can be improved
20
21
PCR demonstration & characterization
22
Coarse filter for debris filtration
Cellular remains
-PCR
DNA
ladder
NC
Only debris filtered
PC
Filtered PCR products
23
The newly developed SNP sensor
Cavity in PDMS (700mm)
Cavity‐electrodes
Au (100nm) on Ti (5nm)
SiNx (100nm) on Si‐substrate
Electrodes
Cavity
PDMS
• Photolithographic process for electrode
• Small and well-defined cavities
realized by PDMS molding.
• Cavity volume: down to 0.5 µL
24
Electrochemical ＳＮＰ sensing
Electrochemical sensing of allele specific PCR products
Chemical reaction
SNP
TAQ PPi
H. Tanaka, Jpn. J. Appl. Phys. 51 (2012) 04DL02.
(Pyloric acid)
Current detection
[Fe(CN)6]4+
PPi
NAD+
Pyrophosphaze
e-
Diaphoraze
[Fe(CN)6]+3
GAPDH
NADH
eBPG
Pi+GAP
25
ABO gene SNP detection
Sequences of exon 6 inhuman AB,O gene
O allele
AB allele
Newly designed
allele-specific primer
26
The newly developed SNP sensor
Detector is tested by using a ferricyanide solution.
Molarity is similar to the one which will be obtained in real case later.
Current(uA)
current
(uA)
2
1
Imax
0
-1
-2
0.5 uL
10.0 uL
-0.4
0.4
Voltage (V)
2.0
PeakCurrent(uA)
current (uA)
Cyclic voltammetric
measurements for
various detector volumes
1.5
10 uL
3 uL
1.0
0.5
0.0
0.0
0.5 uL
1.0 uL
0.4
0.8
1.2
2
Detector
area
Area
(mm(mm
) 2)
27
SNP detection results
CYP2C9
(warfarin)
K-ras
(cetuximab)
CYP2D6
(tamoxifen)
DNA-ladder
D Current (nA)
M
W
M
M
W
W
600
400
200
M
M
W
W
W
M
0
M: mutant = SNP exist
W: wild = no SNP
28
SNP Detection chip
DNA amp.
（PCR）
DNA
extraction
Pump
DNA
purification
detection
SNP: A
Microfluidic chip
Buffer
SNP: B
Pump
Valve
SNP: C
Mixing
SNP: D
DNA filter
PCR
Blood
1uL
SNP: E
Pump
SNP sensing
Rapid genotyping diagnostic on chip system
• Automated DNA extraction from blood
• Output diagnostic results within 2 hours
The newly developed DNA filter
29
The highly precise structure for high resolution DNA filter
→
• DNA separation length reaches less than 50base pair.
• The separation can be achieve in several seconds.
Silicon columns
DNA separation by IP-RP HLC
(ion-pair reverse phase high performance liquid chromatography)
The newly developed DNA filter
DNAs are filtered through the Si pillar forest
DNAs
DNAseparation
Silicon Pillar Array
Φ1m Height 30m
Interval 1m
The shorter, the faster DNAs go
through the filter.
30
31
The newly developed DNA filter
DNA separation on chip based on Ion-pair reversed phase chromatography
 With gradient mobile phase (concentration is changing during filtering):
100 bp
150 bp
250 bp
500 bp
Intensity (A.U.)
0.90
0.50
DNA diluent
0.13
Time (min)
0
5
10
20
15
25
30
Clear separation and results are reproducible.
50 bp ladder an 10 bp ladder are separated on chip.
Full automatic SNP detection LoC
32
Outsourcing
Service
Present：manual handling )
DNA
extraction
DNA amp.
（PCR）
DNA
purification
Large Equipments
detection
（several
business days）
SNP
judgment
Our target is SNP-detection from blood
around 1 hour with small one-chip.