DNA amplification George Kokkoris, g.kokkoris@inn.demokritos.gr, - - PowerPoint PPT Presentation

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification

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Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification

National Center for Scientific Research ‘Demokritos’ Institute of Microelectronics Athens, Greece George Kokkoris, g.kokkoris@inn.demokritos.gr, 2106503238

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification

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• Introduction
• DNA amplification by polymerase chain reaction (PCR)
• Miniaturized or μ-PCR devices
• The continuous flow μ-PCR device and its fabrication
• Modeling heat transfer in the μ-PCR device
• The unit cell & mathematical formulation
• Results
• Temperature distribution in the μ-PCR device
• Power requirements

Contents

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification

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DNA amplification by polymerase chain reaction

• Polymerase chain reaction (PCR)
• can create copies of specific fragments of DNA by cycling through three

temperature steps: denaturation at 367 – 371 K, annealing at 323 – 338 K, and extension at 348 – 353 K.

denaturation 368 K (95 oC)

Each thermal cycle can double the amount of DNA, and 20–35 cycles can produce millions of DNA copies

annealing 333 K (60 oC) extension 350 K (77 oC)

• by the amplification allows the detection, of very small amount, of traces of DNA in

the starting material; this is very important in forensic analysis and medical diagnosis. A cycle:

H-bonds disrupt primers catalyzed by DNA (e.g. Taq) polymerase

1#2

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification

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DNA amplification by polymerase chain reaction

2#2

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification

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Devices for PCR

• Conventional (large scale) thermal cyclers

1#7

[B. Παπαδόπουλος, “Συγκριτική υπολογιστική μελέτη μικρορευστονικών διατάξεων για την ενίσχυση δειγμάτων DNA μέσω της αλυσιδωτής αντίδρασης πολυμεράσης”, διπλωματική εργασία (2015)]

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification

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Devices for PCR

• Miniaturized PCR (μ-PCR) devices

continuous flow static chamber or batch fixed loop closed loop miniaturized thermocyclers natural convection based 2#7

• scillatory

droplet based

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification

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Devices for PCR

• Static chamber or batch μ-PCR devices

miniaturized thermocyclers natural convection based 3#7

[Shen et al., Sens. And Actuators B (2005)]

• Both the sample and the

device undergo thermal cycling

• Fast transitions from one

temperature level to the

• ther are required

[Priye et al., Analytical Chemistry (2013)]

• Constant temperature at

~100oC at the cylinder bottom and ~50oC at the cylinder top

• Natural convection due to

temperature gradient

• Full flexibility on the

PCR protocol (i.e. the number of cycles and the duration may vary)

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification

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Devices for PCR

• Continuous flow μ-PCR devices

fixed loop 4#7

[Kopp et al., Science (1998)]

• Only the sample undergoes thermal

cycling

• The number of cycles and the relative

residence time at each thermal zone is defined at the fabrication step

• 2 or 3 temperature levels

droplet based

water droplets in oil carrier-fluid

[Morh et al., Microfluid Nanofluid (2007)]

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification

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Devices for PCR

• Continuous flow μ-PCR devices

closed loop 5#7

• Only the sample undergoes thermal cycling
• Flexibility on the number of cycles
• Takes advantage of buoyancy forces to continuously circulate reagents in a closed

loop through the thermal zones

• The heating required is advantageously used to induce fluid motion without the need

for a pump.

[Chen et al., Anal. Chem. (2004)]

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 10

Devices for PCR

μ-PCR devices 6#7

• scillatory
• Only the sample undergoes thermal

cycling

• Flexibility on the number of cycles
• The microchannel terminates in a

chamber with an enclosed air volume. Upon pumping the liquid through the channel towards the reservoir, the air in the chamber gets compressed. Upon releasing the pump pressure, the air in the chamber expands again, pushing the liquid column back to its original position

• Main drawback is the increased

complexity of the liquid control.

[Becker et al., Proc. of SPIE Vol. 8976 (2014)]

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 11

Devices for PCR

• The development of μ-PCR devices shares the

motivation for μTAS (micro-Total Analysis Systems), i.e.

• faster process [for PCR 1-2 h (thermal cycler), 4-15 mins (μ-PCR)]
• reduced power consumption
• decreased cost for fabrication and use: disposability
• portability
• smaller sample size

Towards point of care (diagnostic) devices

Schematic of a fixed loop, continuous flow μ-PCR

[Kopp et al., Science (1998)]

water droplets in oil carrier-fluid from a droplet based μ-PCR

[Morh et al., Microfluid Nanofluid (2007)]

heater heater heater

• Substrate materials
• μ-PCR devices were fabricated on Si, then on glass,

and later on polymeric substrates (low cost, biocompatibility, flexibility)

Schematic of a closed loop, continuous flow μ-PCR

[Chen et al., Anal. Chem. (2004)]

7#7

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 12

Example 1: Temperature uniformity in continuous flow μPCR

• Fabrication steps

& modeling

Schematic of a fixed loop, continuous flow μ-PCR [Kopp et al., Science (1998)]

• A continuous flow, fixed loop, μ-PCR device with

integrated heaters, fabricated on PI (polyimide) substrate power requirements (cost, portability) temperature distribution in the device (the temperature control of the DNA sample is crucial for the efficiency of amplification)

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 13

The microfluidic device

• Resistive Cu heaters (top-down view
• f the bottom side)
• Cross section of the channel
• Microfluidic channel (top-down view
• f the top side)

μ 50 μm 50 μm 30 μm 70 μm

PDMS: poly(dimethyl)siloxane

PE: polyethylene PI: polyimide

channel

Cu Cu

20 μm

heaters

150 μm 100 μm 100 μm

25 thermal cycles, total length: ~2 m

~ 5.7 cm ~ 2.7 cm

Heater 1, 368 K (denaturation) Heater 2, 350 K (extension) Heater 3, 333K (annealing)

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 14

• 1. Double-sided Cu-clad polyimide

(Pyralux™)

The fabrication steps

PDMS PE PI Cu Cu

• 2. Coating with AZ photoresist
• 3. AZ UV exposure and

development PI Cu Cu AZ PI Cu Cu

AZ AZ

• 4. Cu wet etching and removal
• f AZ

PI Cu

Cu Cu

• 5. Coating with AZ photoresist

PI Cu

Cu Cu

AZ

• 6. AZ UV exposure and

development PI Cu

Cu Cu

AZ AZ

• 7. Cu wet etching and removal of AZ

PI

Cu Cu

Cu Cu

• 8. Plasma etching of PI and

removal of Cu PI

Cu Cu

PI

Cu Cu

• 9. Channel sealing (lamination)

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 15

Device testing

Microfluidic Silica tube Silica tube

• Flow test for leakages. Filling with a red dye

Syringe pump The microfluidic channel filled with red dye

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 16

• The unit cell of the microfluidic device

Modeling: The unit cell used for the calculations

Heater 2, 350 K (extension) Heater 3, 333K (annealing)

25 thermal cycles, total length: ~2 m

~ 5.7 cm ~ 2.7 cm Heater 1, 368 K (denaturation)

• 1 thermal cycle is performed at the unit cell
• The aim is to calculate the temperature in the channel

(denaturation) (extension) (annealing)

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 17

The mathematical formulation

• Heat transfer in the moving DNA

sample (steady state)

• Heat transfer in all polymeric blocks,

i.e. PI, PE, PDMS (steady state)

• Heat transfer in resistive heaters (steady state)

BC: Periodic boundary conditions @ yz boundaries, inlet, and outlet boundaries, and convective cooling at the rest of the boundaries, continuity conditions at internal interfaces.

• Momentum conservation and continuity equations for the flow in the channel (steady state)

BC: uniform velocity profile @ inlet, no slip condition at the channel walls, pressure & no viscous stress at the outlet T is the temperature, k the thermal conductivity, ρ the density, Cp the specific heat, and u the fluid velocity vector, g is the acceleration of gravity, p is the pressure, μ the dynamic viscosity of the fluid (fluid: DNA sample), is the rate of heat generation per unit volume at a resistive heater.

     ( )

p

k T C T u

   ( ) k T

         

2

p u u g u

( ) k T g   

g

Periodic BC Periodic BC

[Numerical solution: COMSOL]

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 18

uin=0.001 m/s uin=0.01 m/s

Results: Temperature distribution in the device

• Uniform temperature distribution above the heaters
• Not important thermal “cross talk”

Heater 2 Heater 3 Heater 1 Heater 2 Heater 3 Heater 1

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 19

Results: Temperature profile along the channel axis

• The temperature uniformity is better and changes are steeper for the low u case

uin=0.001 m/s uin=0.01 m/s

Conditions: 0.001 m/s, Q1=19 mW, Q2=15 mW, Q3= 8 mW / 0.01 m/s, Q1=20 mW, Q2=15 mW, Q3= 6 mW

‧ ‧ ‧ ‧ ‧ ‧

denaturation annealing extension (denaturation) (extension) (annealing)

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 20

Results: Measures of performance

• The temperature uniformity in a channel zone (denaturation, annealing,

extension) is expressed by the percentage of the zone length with temperature in the acceptable range (3 K).

• power requirements at the heaters (Qk, k=1,2,3)

.

uin=0.001 m/s uin=0.01 m/s Power for the unit cell Temperature uniformity in the zones

denaturation zone annealing zone extension zone

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 21

Results: Current requirements at the resistive heaters

 Q I R  L R ρ A

• According to Joule’s first law, the current we have to apply at the resistive heaters is

where ρ is the electrical resistivity, L is the length of resistive line, and A is the cross-sectional area of the line

,

• Cu is the material of the resistive heaters (lines)

Power for the unit cell Current requirements @ heaters

uin=0.001 m/s uin=0.01 m/s

Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 22

Results: The effect of the device material

• Much better uniformity and lower power requirements

for the PI based device

• Si device requires power an order of magnitude greater for

heater 1 and cooler instead of heater 3 PI-PDMS-PE Si glass

Conditions: u=0.001 m/s

• Temperature profile along the channel axis for PI, Si, and glass based devices

Temperature uniformity Power for the unit cell

denaturation zone annealing zone extension zone

denaturation annealing extension