Presentation on the latest research on "Microfluidic PCR Devices for DNA Amplification". Might be helpful for students and others who are interested. Report Included

1. Microfluidic PCR Devices
for DNA Amplification
Submitted by: Farid MUSA
BioMEMS - Fabrication Technologies and Applications (BE544)
Izmir Institute of Technology, Department of Bioengineering

2. OUTLINES
 Brief Introduction to Polymerase Chain Reaction (PCR)
 Why PCR in Microfluidic Devices?
 Advantages and Drawback
 Brief History
 Current Applications and Outlook
 Device Materials and Fabrication Methods
 Design Approaches for Microfluidic PCR Devices
 Conclusion
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3. Brief Introduction to Polymerase Chain Reaction (PCR)
 Polymerase Chain Reaction (PCR) is a DNA amplification technique used to increase
(or amplify) the number of DNA fragments.
 PCR was invented by Kary Mullis in the 1980s.
 PCR is the most common DNA amplification method in molecular biology and
forensic science.
 Alternative methods are isothermal amplification techniques such as NASBA, LDR,
RCA, etc.
 In short, PCR process can be divided into three distinct steps. Each step is
performed at different temperature and amplification is carried out by cyclic
repetitions.
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4. Brief Introduction to Polymerase Chain Reaction (PCR)
 Initial solution contains:
 DNA template (sample DNA that contains the target sequence)
 Taq DNA polymerase (derived from thermophile organisms “thermos
aquaticus”)
 DNA Primers
 Nucleotides (dNTPs)
 Thermal Stages in PCR
 Denaturation (≈95°C)
 Double stranded DNA denaturates into two single strands
 Annealing(≈56°C)
 Primers attach to single stranded target DNA
 Extension(≈72°C)
 DNA polymerase activates and synthesizes complementary strand of DNA
from free nucleotides in solution
 Thermal cycling results in exponential increase of double
stranded DNA concentration.
Annealing
≈56°C
Extension
≈72°C
Denaturation
≈95°C
Refs: (Ahrberg et al., 2016) 4

5. Microfluidics Devices for PCR
(Advantages and Drawback)
 Advantages over conventional PCR devices
 Negligible thermal inertial effects due to small thermal mass
 Controllable rapid heat and mass transfer due to large surface-to-volume ratio
 Reduced sample and reagent consumption due to smaller volumes
 Small but sufficiently large volume for application of bulk kinetics
 Inexpensive operation of the systems
 Portable and disposable devices
 Typical Drawbacks
 Adsorption of PCR mixture on flow channel surface causes PCR inhibition and carryover
contamination
Refs: (Zhang et al., 2016), (Zhang et al., 2006) 5

6. Microfluidics Devices for PCR
(Brief History)
Year Event
1975
First miniaturized gas chromatograph fabricated on a single silicon wafer at Stanford
University
1993 First silicon-based stationary PCR chip was described
1996 First PCR-CE(Capillary Electrophoresis) integrated microfluidics device was presented
1998 First continuous-flow PCR on a chip
2000 Integration of PCR with DNA microarray hybridization on a monolithic chip
… etc.
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7. Microfluidics Devices for PCR
(Current Applications and Outlook)
 Current Applications
 Today some commercial thermal cyclers and real-time thermal cyclers have integrated microfluidic
PCR devices.
 Quantitative PCR (qPCR) or Real Time PCR – used in quantification of the target molecules during
measurement of real time amplicons
 Digital PCR (dPCR) - Absolute quantification of the target molecules
 Future Perspective
 Droplet-based continuous-flow microfluidic PCR
 PCR Technologies for Point of Care Testing
 Smart droplet-based microfluidic platform for autonomous scientific discovery
Refs: (Ahrberg et al., 2016), (Petralia et al., 2017), (Zhang et al., 2016) 7

8. Device Materials and Fabrication Methods
 Materials used in non-chip PCR microfluidics are polytetrafluoroethylene (PTFE),
fused-silica, borosilicate glass block, etc.
 Most common materials used in PCR microfluidics chip devices are:
 Silicon
 Glass
 Polydimethylsiloxane (PDMS)
 Polycarbonate (PC)
 Poly(methyl methacrylate) (PMMA)
 Polyimide (PI)
 Polyethylene terephathalate (PET)
 etc.
 Surface of the material used in PCR microfluidic devices must have minimal surface-
to-molecule interaction in order to reduce PCR inhibition
Refs: (Zhang et al., 2006) 8

9. Device Materials and Fabrication Methods
 Kodzius et al. have studied biocompatibility of PCR mixture and several materials
which are used in PCR microfluidic devices
 Interaction was determined by running samples in 4% agarose gel and results were
presented in terms of relative band intensity (RBI)
 Particularly material interactions with following were studied:
 PCR mixture with and without BSA (Bovine Serum Albumin)
 DNA template
 DNA polymerase
 According to results
 Least PCR inhibitory plastic material were PP, PTFE, and PDM
 Mineral oil, which is important in droplet microfluidics has little influence on PCR
 Metal tubes inhibited PCR by interactions with polymerase rather than DNA template
 Bare silicon was found to be more inhibitory on DNA polymerase than silica
 Presence of BSA in the PCR reaction had significantly improved surface compatibility of
most materials
Refs: (Kodzius et al., 2012) 9

10. Device Materials and Fabrication Methods
Refs: (Kodzius et al., 2012) 10
PCR inhibition through material interaction with template DNA or the polymerase correspondingly.

11. Device Materials and Fabrication Methods
 Fabrication of PCR microfluidic device from silicon and glass include most of the
silicon micromachining technology such as photolithography, thermal oxidation,
etching, vapor deposition, bonding techniques, etc. and novel techniques such as
shadow mask.
 Common polymer fabrication methods include
 Replication methods such as hot embossing, injection molding, casting, soft lithography, etc.
 Direct fabrication methods such as laser ablation, plasma etching, X-ray lithography, stereo-
lithography, SU-8, LIGA, etc.
Refs: (Zhang et al., 2006) 11

12. Design Approaches for Microfluidic PCR Devices
Refs: (Ahrberg et al., 2016)
Designs approaches for microfluidic PCR devices
 Position dependent temperature variation (Continuous Flow)
 Serpentine channel design
 Radial design with droplet microfluidics
 Digital and oscillating design
 Time dependent temperature variation (Stationary Chamber)
 Stationary single(or multiple) chamber devices
 Droplet based digital PCR
 Centrifugal microfluidic design
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13. Design Approaches for Microfluidic PCR Devices
Refs: (Moschou et al., 2014)
Type Serpentine channel design (Continuous)
Studied by Moschou et al. in 2014
Number of thermal cycles 30 per chip
Materials used Flexible PI substrate
Heating system Copper microheaters
Channel width • 400 µm for extension zone
• 200 µm for the denaturation and annealing zones
Advantages • Amplification time for 30 cycles ≈5 min
• Low power consumption ≈2.4 W
• Disposable chip
• Can be integrated into complex LoC systems
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14. Design Approaches for Microfluidic PCR Devices
µPCR chip fabrication process flow. Fabricated PCR device
Refs: (Moschou et al., 2014) 14

15. Design Approaches for Microfluidic PCR Devices
The volume % of each zone in the acceptable
temperature range (±1.5 K) vs. the volumetric flow rate.
The pressure drop in a unit cell vs.
the volumetric flow rate
Refs: (Moschou et al., 2014) 15

16. Design Approaches for Microfluidic PCR Devices
Refs: (Manage et al., 2011)
Type Stationary single chamber device (Stationary)
Studied by Manage et al. in 2011
Number of thermal cycles 35 cycles
Materials used Flexible PDMS layer sandwiched between Borofloat glass
substrates
Heating system Peltier element
Chamber Depth 90 µm (PCR chamber volume is 600 nL)
Advantages • Amplification time for 35 cycles ≈40 min
• Whole blood sample as template
• Directly amplification of viral and genomic DNA
templates
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17. Design Approaches for Microfluidic PCR Devices
(a) Schematic of the PCR/CE glass/PDMS/glass microfluidic chip and (b) photograph of the chip
Refs: (Manage et al., 2011) 17

18. Design Approaches for Microfluidic PCR Devices
Why Droplet Microfluidics for PCR?
 Solutions can be divided into millions of small droplets
 Each droplet act as independent PCR reaction chamber
 Individual sample is encapsulated in single droplet; hence, PCR inhibition and carryover
contamination is minimized.
 Possible to encapsulate single DNA molecules in a picolitre and even femtolitre droplets
 Emulsion is most common method for PCR droplet generation
 Dispersed phase is the PCR mixture water solution
 Continuous phase is usually mineral oil for PCR application
18Refs: (Yi-Qiang et al., 2016)

19. Design Approaches for Microfluidic PCR Devices
Refs: (Yolanda et al., 2009)
Type Radial design (Continuous)
Studied by Yolanda et al. in 2009
Number of thermal cycles 34 per chip
Materials used SU-8 embedded in PMMA
Heating system Copper rod and annular Peltier module
Channel width 200-500 µm
Advantages • Amplification time for 34 cycles ≈17 min
• Droplet based
• Can be integrated into complex LoC systems
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20. Design Approaches for Microfluidic PCR Devices
Design of the radial PCR device
Refs: (Yolanda et al., 2009) 20
 Orange region (D) corresponds to
denaturation zone
 Channels travelling to the periphery pass
through annealing and extension zones
 Droplets are introduced at (B1 and B2)
 Oil is introduced at (A)
 Droplets form at T junction (C)
 Amplified samples exit at (F)
 Peltier module is placed under blue and
orange regions

21. Design Approaches for Microfluidic PCR Devices
Temperatures of droplets in
denaturation zone vs. copper rod
temperature
Refs: (Yolanda et al., 2009)
Analysis of PCR products.
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22. Design Approaches for Microfluidic PCR Devices
Refs: (Bian et al., 2015)
Type Droplet based digital PCR (Stationary)
Studied by Bian et al. in 2015
Number of thermal cycles 35 cycles
Materials used PDMS
Reaction Chamber 28 mm in length and 15 mm in width
Advantages • Amplification time for 35 cycles ≈ 40 min
• Single-molecule sensitivity
• Simultaneous detection of E. coli O157: H7 and L. monocytogenes
Drawbacks Total assay time is about 18 hours
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23. Design Approaches for Microfluidic PCR Devices
Relationship between the measured value (copies/μL) in the duplex ddPCR
assay and the expected value by absorbance detection 260 nm
Refs: (Bian et al., 2015)
Mineral oil saturated PDMS (OSP) microfluidic chip.
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24. Conclusion
 Slowly microfluidic PCR chips or integrated devices are dominating
PCR market
 Currently variety of design approaches are present for microfluidic
PCR chips
 In general, continuous microfluidic PCR are more efficient than
stationary chamber PCR
 Material of PCR microchannels or chambers have significant effect
on the process efficiency
 Emulsion droplet microfluidic has the potential to increase PCR
efficiency and eliminate problems such as PCR inhibition and
carryover contamination
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25. References
 Zhang, Y., & Jiang, H. R. (2016). A review on continuous-flow microfluidic PCR in droplets: Advances, challenges and future. Analytica
chimica acta, 914, 7-16.
 Manage, D. P., Morrissey, Y. C., Stickel, A. J., Lauzon, J., Atrazhev, A., Acker, J. P., & Pilarski, L. M. (2011). On-chip PCR amplification of
genomic and viral templates in unprocessed whole blood. Microfluidics and nanofluidics, 10(3), 697-702.
 Zhang, C., Xu, J., Ma, W., & Zheng, W. (2006). PCR microfluidic devices for DNA amplification. Biotechnology advances, 24(3), 243-284.
 Petralia, S., & Conoci, S. (2017). PCR Technologies for Point of Care Testing: Progress and Perspectives. ACS sensors, 2(7), 876-891.
 Ahrberg, C. D., Manz, A., & Chung, B. G. (2016). Polymerase chain reaction in microfluidic devices. Lab on a Chip, 16(20), 3866-3884.
 Kodzius, R., Xiao, K., Wu, J., Yi, X., Gong, X., Foulds, I. G., & Wen, W. (2012). Inhibitory effect of common microfluidic materials on
PCR outcome. Sensors and Actuators B: Chemical, 161(1), 349-358.
 Moschou, D., Vourdas, N., Kokkoris, G., Papadakis, G., Parthenios, J., Chatzandroulis, S., & Tserepi, A. (2014). All-plastic, low-power,
disposable, continuous-flow PCR chip with integrated microheaters for rapid DNA amplification. Sensors and Actuators B: Chemical, 199,
470-478.
 Schaerli, Y., Wootton, R. C., Robinson, T., Stein, V., Dunsby, C., Neil, M. A., ... & Hollfelder, F. (2008). Continuous-flow polymerase chain
reaction of single-copy DNA in microfluidic microdroplets. Analytical chemistry, 81(1), 302-306.
 Yi-Qiang, F. A. N., Mei, W. A. N. G., Feng, G. A. O., ZHUANG, J., Gang, T. A. N. G., & ZHANG, Y. J. (2016). Recent development of
droplet microfluidics in digital polymerase chain reaction. Chinese Journal of Analytical Chemistry, 44(8), 1300-1307.
 Bian, X., Jing, F., Li, G., Fan, X., Jia, C., Zhou, H., ... & Zhao, J. (2015). A microfluidic droplet digital PCR for simultaneous detection of
pathogenic Escherichia coli O157 and Listeria monocytogenes. Biosensors and Bioelectronics, 74, 770-777.
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26. Questions?
THANKS FOR ATTENTION
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