A Lab-on-Chip System for direct SNP sensing from human blood What is SNP

1

A Lab-on-Chip System for direct SNP sensing from human blood

1Advanced Technology Research Laboratory, Panasonic corp. 3-4 Hikaridai, Seika-cho, Soraku-gun, Kyoto 619-0237, Japan

2Imec, Kapeldleef 75, B-3001, Leuven, Belgium3Nara Institute of Science and Technology, 8916-5 Takayama-cho Ikoma

Ichiro Yamashita1,3, Paolo Fiorini2

22

Single Nucleotide Polymorphism

One DNA alternation can affect people’s sensibility against drugs

T C A G G T C C G C

A G T C C A G G C G

T C A G G C C C G C

A G T C C G G G C G

ＳＮＰpredisposition to diseases, difference in reaction on drugs (ie. adverse side effects), etc

What is SNP

33

No SNP exist SNP exists

Electrode

A

enzymatic reaction

Target DNA froma patient

enzymatic reaction

e-

Current

phosphate compound

replication No replication

Target DNA fixation

is not necessaryPatient

DNA

SNP sensor by Panasonic

44

Our target is SNP-detection from bloodless than 1 hour with small one-chip.

DNAextraction

DNA amp.（PCR）

detection

Large Equipments

DNA purification

Present：manual handling )

Outsourcing Service（several

business days）

SNPjudgment

Easy and fast detection of SNP

55

Buffer

SNP sensing

Mixing

Pump

Pump

Pump

DNA filter

Valve

Blood1uL

SNP: A

SNP: B

SNP: C

PCR

SNP: D

SNP: E

Microfluidic chip

DNAextraction

DNA amp.（PCR） detection

DNA purification

Rapid genotyping diagnostic on chip system• Automated DNA extraction from blood • Output diagnostic results within 1 hour

SNP Detection chip

66

1cm

Buffer

SNP sensing

Mixing

Pump

Pump

Pump

DNA filter

Valve

Blood1uL

SNP: A

SNP: B

SNP: C

PCR

SNP: D

SNP: E

SNP Detection chip

77

Blood

Drain Drain

DetectorPCR 1 PCR 2

Reagent 1

Reagent 2

Mixer 2

Pump

Coarsefilter

Mixer 1

Valve Valve

Module 1 Module 2a

Sampleloading

DNA extractionand amplification

Allele specificamplification

Electro‐chemical  detection

Single SNP Detection chip details

88

8

Pump

Valve

Detector

PCR

Single SNP Detection Chip

99

PCR 1Mixer 1 Coarse filter

PCR 2Mixer 2

Pump Valve

Detector

Single SNP Detection device

Blood

Drain Drain

DetectorPCR 1 PCR 2

Reagent 1

Reagent 2

Mixer 2

Pump

Coarsefilter

Mixer 1

Valve Valve

Module 1 Module 2a

1010

Thermal solution

ReservoirValve ValvePump

Mixer

PCR

Silicon layerPlastic layer

Pyrex layer

PCR 1Mixer 1 Coarse filter

PCR 2Mixer 2

Pump Valve

Detector

Single SNP Detection device

11Fabrication SSD (Process flow)

11

1 µm therm oxide

Blanket Si wafers

BS oxide removal wet

IX-litho

Oxide etch

Si etch, Strip

Anodic bonding

Pyrex wafer

Grinding and cleaning

Litho

Etch and strip

11

12Fabrication SSD (Results)

12

Coarse filter

FS looking through glass

BS Si surface

Coarse filter

Mixer

PCR chamber

~300µm

12

13

Silicon sub.

13

• Operation pressure 3MPa ・1.5V battery drive

• Maximum generation pressure 30MPa

case

+ -+-Layered polymer actuator

electrode electrode

Diaphragm

Pumping action

The newly developed pump unit

1414

ElectrolyteEMI-TFSI +-

sewell

shrink

BatteryElectric power

V

－

－－

－

－

－

－

－

－

－+

+ +

++

Distortionup to 10％ OK

Reduce

Oxid

Polymer

Polymer:

polypyrrole (PPy) base with TFSI

Operating principle：volume change by injection/ extraction of electrolyte ions

Conductive Polymer Actuator

EMI

NN NN

－

+

NN NN

－＋

reduction

DEMI+ > DTFSI-

D: diffusion constant

EMI+

PPy-TFSI (charge: neutral)

negative charge attracts positive ions from electrolyte.

Py+ Py0NN

1515

0

1

2

3

50

Str

ain

(%)

0 100

Time (s)

Load [MPa]0.51.02.07.0

10.0

15.0

1cm

Polymer pump characterization

active CPmetal

whole Glue

dot Glueinactive CP

Adhesive dots

16

Mixers : characterization

Mixing of two fluids

Mixing of particles (simulating blood cells)

inlet

outlet

inlet

Inlet Port 1

Outlet Port

Inlet Port 2

1717

PCR chamber part of the LoC PCR chamberFilled 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

PCR demonstration & characterization

18

On chip PCR: fabrication and thermal tests

Thermal insulation is further improved by winding in/out channels around the cavity.

x

z

AA

Section View A-A

Liquid

Cover Glass

Silicon Walls

Thermal Isolation Gap (Air)

-20

-10

0

10

20

30

40

50

60

70

80

-1.2 -0.8 -0.4 0 0.4 0.8 1.2Te

mp

era

ture

Dif

fere

nce

, T

-Ta

mb

(K)

TEC Current (A)

PCR Temp.

Chip Temp.

Steady‐state temperature of the PCR chamber and bulk silicon substrate.

Max 10 K above RT

18

1919

20

30

40

50

60

70

80

90

100

0 5 10 15 20

Temperature (°C)

Time (min)

60

65

70

75

80

85

90

95

100

0 20 40 60 80 100 120

Temperature (°C)

Time (s)

Micro-PCRresult

Template: Whole Blood

Successful micro-PCR amplification in less than 21 minutes -> 11 minutes.

Fast Micro-PCR Demonstration

20

Template: Human Genomic DNA(From Commercial Source)

DN

AL

add

er

Po

siti

veC

on

tro

l

Neg

ativ

eC

on

tro

l

Mic

ro-

PC

R

Protocol2X buffer KOD fx 5 L2 mM dNTP’s  1 L10 M Fw primer 1 L10 M Rv primer 1 LKOD polymerase 0.4 LWater 1.1 LTemplate 0.5 LTotal 10 L

(2 L amplifiedin micro‐PCR)

T anneal = 60°C# Cycles = 30

Test chip is preloaded with PCR buffer solution

Successful amplification of target fragment in micro-PCR

Reproducibility can be improved

SSD Amplification Tests (Human Genomic DNA)

Commercial Tool SSD

20

2121

PCR demonstration & characterization

2222

DNAladder

PC

NC

-P

CR

Filtered PCR products

Coarse filter for debris filtration

Only debris filtered

Cellular remains

2323

• Photolithographic process for electrode • Small and well-defined cavities

realized by PDMS molding.• Cavity volume: down to 0.5 µL

PDMS Cavity

Cavity in PDMS (700mm)

SiNx (100nm) on Si‐substrate

Cavity‐electrodesAu (100nm) on Ti (5nm)

Electrodes

The newly developed SNP sensor

24

Electrochemical sensing of allele specific PCR products

TAQ 

Chemical reactionSNP

Electrochemical ＳＮＰ sensing

Pi+GAP

NAD+

NADHBPG

[Fe(CN)6]4+

[Fe(CN)6]+3

GAPDHDiaphoraze e-Pyrophosphaze

e-

PPiCurrent detection

PPi(Pyloric acid)

H. Tanaka, Jpn. J. Appl. Phys. 51 (2012) 04DL02.

2525

AB allele

ABO gene SNP detection

Sequences of exon 6 inhuman AB,O gene

O allele

Newly designed allele-specific primer

2626

Detector is tested by using a ferricyanide solution. Molarity is similar to the one which will be obtained in real case later.

2.0

1.5

1.0

0.5

0.0

Cu

rre

nt(

uA

)

1.20.80.40.0

Area (mm2)

Pea

k cu

rren

t (u

A)

Cyclic voltammetric measurements for

various detector volumes

2

1

0

-1

-2

Cu

rre

nt(

uA

)

-0.4 0.4Voltage (V)

0.5 uL 10.0 uL

Imax

0.5 uL1.0 uL

3 uL

10 uL

Detector area (mm2)

curr

ent

(uA

)

The newly developed SNP sensor

2727

0

200

400

600

DC

urr

ent

(nA

)

CYP2C9(warfarin)

CYP2D6(tamoxifen)

K-ras(cetuximab)

DNA-ladder

MM

M

M M

M

W WW

W WW

SNP detection results

M: mutant = SNP existW: wild = no SNP

2828

Buffer

SNP sensing

Mixing

Pump

Pump

Pump

DNA filter

Valve

Blood1uL

SNP: A

SNP: B

SNP: C

PCR

SNP: D

SNP: E

Microfluidic chip

DNAextraction

DNA amp.（PCR） detection

DNA purification

Rapid genotyping diagnostic on chip system• Automated DNA extraction from blood • Output diagnostic results within 2 hours

SNP Detection chip

29

DNA separation by IP-RP HLC (ion-pair reverse phase high performance liquid chromatography)

Silicon columns

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.

The newly developed DNA filter29

30

DNAseparation

DNAs

Silicon Pillar Array

Φ1m Height 30m Interval 1m

The shorter, the faster DNAs go through the filter.

DNAs are filtered through the Si pillar forest

The newly developed DNA filter30

3131

DNA separation on chip based on Ion-pair reversed phase chromatography

With gradient mobile phase (concentration is changing during filtering):

Clear separation and results are reproducible.

50 bp ladder an 10 bp ladder are separated on chip.

0 5 10 15 20 25 30

0.90

0.50

0.13Time (min)

Intensity (A.U.)

100 bp 150 bp 250 bp 500 bp

DNA diluent

The newly developed DNA filter

3232

Our target is SNP-detection from bloodaround 1 hour with small one-chip.

DNAextraction

DNA amp.（PCR）

detection

Large Equipments

DNA purification

Present：manual handling )

Outsourcing Service（several

business days）

SNPjudgment

Full automatic SNP detection LoC