Lab-on-a-Chip for Studying Growing Pollen Tubes - Plant Cell Morphogenesis: Methods and Protocols - page 229

2 Materials

1. Computer-aided design (CAD) drawing software ( see Note 1 ).
2. Fluid-fl ow simulation software ( see Note 2 ).
3. A class 1000 cleanroom facility ( see Note 3 ).
4. Silicon wafers (WRS materials) ( see Note 4 ).
5. Sulfuric acid (H 2 SO 4 ), peroxide (H 2 O 2 ), and a glass container.

6. Hydrofl uoric acid (HF), HF antidote (calcium gluconate gel),
sodium bicarbonate, and a Tefl on container.
7. Negative photoresist SU-8 2035 (MicroChem).
8. SU-8 developer (MicroChem).
9. Isopropyl alcohol (IPA).
10. Deionized water.
11. Hot plate.
12. UV light exposure system.
13. Trichlorosilane.
14. Polydimethylsiloxane (PDMS) (Sylgard ® 184 Silicone
Elastomer Kit—base and curing agent).
15. Vacuum desiccator.
16. Cutter, revolving punch, syringes.
17. Plasma cleaner.
18. PVC tubes (peristaltic pump tubing).
19. Camellia japonica pollen.
20. Growth medium: 1.62 mM H 3 BO 3 , 2.54 mM Ca(NO 3 ) 2

⋅ 4H 2 O,

0.81 mM MgSO 4 ⋅ 7H 2 O, 1 mM KNO 3 , 8 % sucrose (w/v), in

distilled water.
21. Microscope with image capture.

3 Methods

Carry out all procedures in the cleanroom at room temperature
(unless otherwise indicated). Meticulously follow all waste disposal
regulations.
1. Design the microfl uidic network according to the intended
application. Here we develop a microfl uidic chip to investigate
the instantaneous growth rate of pollen tubes as they encounter
a mechanical obstacle consisting of a fl at surface oriented at a
defi ned angle relative to the growth direction: 0° (perpendicu-
lar to the growth direction), 30°, and 60° (Fig. 1 ; see Note 5 ).
2. Carry out microfl uidic simulations to support and validate the
platform design. Depending on the simulation result, the

3.1 Microfl uidic
Network Design