Biology Reference
In-Depth Information
development by microscope and continue development if nec-
essary ( see Note 13 ).
7. Hard-bake at 150 °C for 10 min to solidify the remaining
photoresist and reduce mechanical stresses in the structure ( see
Note 14 ).
8. Verify the mold under the microscope ( see Note 15 ).
9. Silanize the silicon/SU-8 mold. The mold is exposed to
trichlorosilane (or simply silane) vapors to prevent the PDMS
replica from sticking to the mold. Use protective gear and
handle with care. Silane is highly fl ammable (fl ash point of
87 °C), highly corrosive, and reacts violently with water. Using
a plastic dropper (or syringe), place four drops of silane in a
glass dish close to the silicon wafer. Close the glass dish and
place it on a hot plate at 70 °C to evaporate the silane. Leave
for at least 4 h. Cool at room temperature before opening the
glass dish to allow the vapors to settle ( see Note 16 ).
3.3 PDMS
Microfl uidic Chip
1. Thoroughly mix ten parts of PDMS polymer base with one part
of PDMS curing agent in a disposable container ( see Note 17 ).
2. Pour the PDMS mix onto the silicon/SU-8 mold. Place the
mold/PDMS ensemble in a vacuum desiccator for 15 min to
degas the PDMS and next cure in an oven at 80 °C for 2 h.
3. Carefully excise each PDMS replica from the mold ( see Note 18 ).
4. Punch inlet and outlet ports of the PDMS replica. Rinse with
IPA and air-dry to clean any dirt particle ( see Note 19 ).
5. Bond the PDMS replica to a glass slide to seal the microfl uidic
chip ( see Note 20 ).
6. Insert inlet and outlet PVC tubes from the top of the structure to
obtain the microfl uidic platform shown in Fig. 2b ( see Note 21 ).
3.4 Microfl uidic
Platform Testing
1. Collect, dehydrate, and store Camellia japonica pollen on sil-
ica gel at −20 °C for later use ( see Note 22 ).
2. Prior to experimentation, thaw and rehydrate a few milligrams
of the pollen in humid atmosphere for 1 h ( see Note 23 ).
3. Prepare liquid growth medium ( see Note 24 ).
4. Place the microfl uidic platform under the microscope (or any
other imaging setup).
5. Immerse the pollen grains in 1 ml liquid growth medium.
Agitate gently to mix the suspension; pollen grains are very
sensitive to excess mechanical stress.
6. Using a syringe, carefully inject the pollen suspension through
the PVC tube into the microfl uidic device. Monitor the injec-
tion through the microscope. Figure 3a shows a typical pollen
grain distribution.
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