practice 6:
PREPARATION AND TESTING OF MICROFLUIDIC CHIP
location: Loschmidt Laboratories, Kamenice 5/A13, 2nd floor, room 208
lecturer: Mgr. Tomáš Buryška (bary@mail.muni.cz)
I. WORKFLOW
– preparation microfluidic chip
– basic chip operation
– droplet microfluidics and microscopy
II. MOTIVATION
Microfluidics can be defined as the science and technology manipulating and analysing fluid
flow in sub-millimeter dimensions. It is becoming important technology for many emerging
applications and disciplines, especially in the fields of chemistry, biology and medicine.
Concrete application examples are biosensor devices for molecular diagnostics, polymerase
chain reaction chips, high-throughput screening, controlled drug delivery systems, drug
discovery methods, forensic analysis instruments, and so on (1).
III. THEORETICAL BACKGROUND
Formation of water in oil droplets in microfluidic chip has several benefits when compared
to standard high-throughput technology. Amongst such benefits belong low volume of
reagents consumed, chip modularity, low cost and simple fabrication. When all pros
combined properly one may encounter drop costs of screening millionfold (2, 3).
In this practice students will put hands on microfluidic chip fabrication on their own. During
the lesson they will go through cutting out and assembling a chip applying oxygen plasma
bonding. Prepared chips are to be used for water in oil droplet generation (4). An example
will follow with encapsulation of single E.coli BL21 DE3 cells to droplets. Finally, there will be
observation of single cells in emulsions generated.
IV. DESIGN OF EXPERIMENT
Solutions and reagents:
– Dow Corning Sylgard® 184
– HFE-7500 oil
– Trichloro(1H,1H,2H,2H-perfluorooctyl)silane
– 1.5M NaCl
– deionized distilled H2O
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– E.coli BL21 DE3 or other bacterial cells
– Percoll ®
– isopropanol
Equipment:
– Chemyx Fusion 200 syringe pumps
– gas-tight syringes (various volumes)
– PTFE tubing
– razor
– biopsy puncher
– glass slide
– USB-microscope
Protocol for PDMS chip assembling:
1. using razor carefully cut PDMS with chip out of plate
2. with the help of ruler cut out single chip and place them bottom up
3. punch holes for inlet and outlet holes with biopsy puncher
4. clean glass slide and PDMS chip with isopropanol
5. place chip on the tray and put inside the oxygen plasma
6. run oxygen plasma for 3 minutes at 50% generator power at oxygen flow around 5scsm
at 200 mTorr
7. immediately after oxygen plasma process ends, bond microfluidic chip and glass cover
and press with your fingers; NOTE: try to avoid having bubbles between PDMS and
glass
8. place bonded chip on hot plate at 60°C for another 5 minutes
9. let chip cool down to room temperature
10. flush channels with 1 % silane solution in HFE-7500, after 3 minutes replace by pure
HFE-7500 oil and air
Droplet formation protocol:
1. load syringes with HFE-7500 oil and 150 mM NaCl, 25 % (v/v) Percoll and properly
diluted cells
2. attach PTFE tubing to the syringe and remove any bubbles
3. put syringes into the syringe pumps, lock tight and set proper syringe diameter
4. connect syringe via tubing into the chip
5. set liquid flow – 300 µL.h-1 for oil phase and 30 µL.h-1 for the aqueous phase
6. observe droplet formation under microscope at various magnification
7. verify cell occupation in emulsions on inverted microsope
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V. HOMEWORK
1. Estimate volume in pico-/femto- litres for monodisperse droplet formed at channel
having dimensions 5, 10 and 20 µm, respectively (assume square cross-section forms
spherical droplets of the same diameter).
2. For calculated droplet volumes estimate approximate cell density in x.10y per mL, to
put single cell per droplet. There is approximately 1.108 cells in medium with OD600 0.5.
Cultivated cells have OD600 4.8. In case that grown culture has insufficient density, calculate
the factor for thickening cell media to sufficient level. Account for 10 per cent pipetting
error.
VI. LITERATURE
1. Dittrich, P. S., and Manz, A. (2006) Lab-on-a-chip: microfluidics in drug discovery., Nat.
Rev. Drug Discov. 5, 210–8.
2. Mazutis, L., Gilbert, J., Ung, W. L., Weitz, D. a, Griffiths, A. D., and Heyman, J. a. (2013)
Single-cell analysis and sorting using droplet-based microfluidics., Nat. Protoc. 8, 870–
91.
3. Yin, H., and Marshall, D. (2012) Microfluidics for single cell analysis., Curr. Opin.
Biotechnol. 23, 110–9.
4. Teh, S.-Y., Lin, R., Hung, L.-H., and Lee, A. P. (2008) Droplet microfluidics., Lab Chip, The
Royal Society of Chemistry 8, 198–220.
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