National Center for Scientific Research ‘ Demokritos ’ Institute of Microelectronics Athens, Greece Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification George Kokkoris, g.kokkoris@inn.demokritos.gr, 2106503238 1 Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification
Contents • 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 2 Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification
DNA amplification by polymerase chain reaction 1#2 • 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. extension annealing denaturation 350 K (77 o C) 333 K (60 o C) 368 K (95 o C) A cycle: H-bonds disrupt primers catalyzed by DNA (e.g. Taq) polymerase Each thermal cycle can double the amount of DNA, and 20 – 35 cycles can produce millions of DNA copies • 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. 3 Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification
DNA amplification by polymerase chain reaction 2#2 4 Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification
Devices for PCR 1#7 • Conventional (large scale) thermal cyclers [B. Παπαδόπουλος, “Συγκριτική υπολογιστική μελέτη μικρορευστονικών διατάξεων για την ενίσχυση δειγμάτων DNA μέσω της αλυσιδωτής αντίδρασης πολυμεράσης”, διπλωματική εργασία (2015) ] 5 Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification
Devices for PCR 2#7 • Miniaturized PCR ( μ -PCR) devices static chamber or continuous flow oscillatory batch miniaturized fixed loop thermocyclers natural closed loop convection based droplet based 6 Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification
Devices for PCR 3#7 • Static chamber or batch μ -PCR devices • Both the sample and the device undergo thermal miniaturized cycling thermocyclers • Fast transitions from one temperature level to the • Full flexibility on the other are required PCR protocol (i.e. the [Shen et al., Sens. And Actuators B (2005)] number of cycles and the duration may vary) • Constant temperature at ~100 o C at the cylinder natural bottom and ~50 o C at the convection based cylinder top • Natural convection due to temperature gradient [Priye et al., Analytical Chemistry (2013)] 7 Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification
Devices for PCR 4#7 • Continuous flow μ -PCR devices fixed loop • 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 [Kopp et al., Science (1998)] water droplets in oil carrier-fluid [Morh et al., Microfluid Nanofluid (2007)] 8 Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification
Devices for PCR 5#7 • Continuous flow μ -PCR devices closed loop [Chen et al., Anal. Chem. (2004)] • 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. 9 Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification
Devices for PCR 6#7 oscillatory μ -PCR devices • 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 [Becker et al., Proc. of SPIE Vol. 8976 (2014)] liquid column back to its original position • Main drawback is the increased complexity of the liquid control. Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 10
Devices for PCR 7#7 heater • Substrate materials • μ -PCR devices were fabricated on Si, then on glass, heater and later on polymeric substrates (low cost, heater biocompatibility, flexibility) Schematic of a fixed loop, continuous flow μ -PCR • The development of μ -PCR devices shares the [Kopp et al., Science (1998)] motivation for μ TAS (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 Schematic of a closed loop, Towards point of care (diagnostic) devices continuous flow μ -PCR [Chen et al., Anal. Chem. (2004)] water droplets in oil carrier-fluid from a droplet based μ -PCR [Morh et al., Microfluid Nanofluid (2007)] Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 11
Example 1: Temperature uniformity in continuous flow μ PCR • A continuous flow , fixed loop, μ -PCR device with integrated heaters, fabricated on PI (polyimide) substrate • Fabrication steps & modeling Schematic of a fixed loop, continuous flow μ -PCR temperature distribution in the device (the [Kopp et al., Science (1998)] temperature control of the DNA sample is crucial for the efficiency of amplification) power requirements (cost, portability) Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 12
The microfluidic device • Microfluidic channel (top-down view • Resistive Cu heaters (top-down view of the top side) of the bottom side) ~ 5.7 cm Heater 1, 368 K (denaturation) ~ 2.7 cm Heater 2, 350 K (extension) Heater 3, 333K (annealing) 25 thermal cycles, total length: ~2 m 150 μ m 50 μ m PE: polyethylene 50 μ m PDMS: poly(dimethyl)siloxane • Cross section of the channel channel 30 μ m PI: polyimide 70 μ m heaters Cu Cu 20 μ m μ 100 μ m 100 μ m Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 13
The fabrication steps 1 . Double-sided Cu-clad polyimide 4. Cu wet etching and removal 7. Cu wet etching and removal of AZ (Pyralux ™) of AZ Cu Cu Cu Cu PI PI PI Cu Cu Cu Cu Cu 8. Plasma etching of PI and 2. Coating with AZ photoresist 5. Coating with AZ photoresist removal of Cu AZ Cu Cu PI PI PI Cu Cu Cu Cu Cu AZ 3. AZ UV exposure and 6. AZ UV exposure and 9. Channel sealing (lamination) development development PE Cu AZ AZ PDMS Cu PI PI PI Cu AZ AZ Cu Cu Cu Cu Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 14
Device testing • Flow test for leakages. Filling with a red dye Silica tube Silica tube Microfluidic Syringe pump The microfluidic channel filled with red dye Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 15
Modeling: The unit cell used for the calculations • The unit cell of the microfluidic device ~ 5.7 cm Heater 1, 368 K (denaturation) (denaturation) ~ 2.7 cm Heater 2, 350 K (extension) (extension) (annealing) Heater 3, 333K (annealing) 25 thermal cycles, total length: ~2 m • 1 thermal cycle is performed at the unit cell • The aim is to calculate the temperature in the channel Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 16
The mathematical formulation Heat transfer in the moving DNA Periodic BC 2 u u g p u sample (steady state) ( k T ) C u T p Heat transfer in all polymeric blocks, i.e. PI, PE, PDMS (steady state) ( k T ) 0 Heat transfer in resistive heaters (steady state) Periodic BC ( k T ) g 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, C p 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 g heat generation per unit volume at a resistive heater. [Numerical solution: COMSOL] Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification 17
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