11. Develop the microstructures cross-linked on the photoresist,
by soaking the wafer into 10 mL of PGMEA twice for 10 min.
12. Wash extensively the wafer with isopropyl alcohol, using a
venting wash bottle, until all the whitish deposits disappear.
13. To make the microstructures more compact and robust, hard
bake the wafer on a hot plate at 180
C for 2 h.
14. Transfer the processed silicon wafer inside a vacuum chamber,
and dispense 200 μL of PFOCTS on a glass slide next to the
wafer. Apply the vacuum overnight to deposit a thin layer of
silanizing agent onto the surface of the wafer.
3.6 Soft Lithography
and Microfabrication
1. Secure the SU8-micropatterned silicon wafer to the bottom of
a square petri dish using fast-drying glue (see Note 21).
2. Pour 120 g of Sylgard 184 prepolymer into a plastic goblet and
add 12 g of Sylgard 184 curing agent base, for making the
PDMS mix.
3. Use a disposable L-shaped spreader to mix the reagents to a
milky consistency until the curing agent is evenly distributed
and the mixture appears full of air bubbles.
4. Place the goblet in the vacuum chamber and apply vacuum. As
the vacuum builds up, bubbles will surface. Vent the chamber a
few times initially to burst the bubbles and then leave it for
complete degassing for 15–30 min.
5. Pour the degassed PDMS mix onto the micropatterned silicon
wafer contained in the petri dish.
6. Place the petri dish in the vacuum chamber and apply vacuum.
As the vacuum builds up, a few bubbles will surface. Vent the
chamber a few times initially to burst the bubbles and then
leave it for complete degassing for 15–30 min.
7. Remove the petri dish and incubate in an oven at 80
C overnight to crosslink the PDMS.
8. Using a scalpel cut gently the PDMS block along the wafer
perimeter, peel off the PDMS block corresponding to the wafer
area, and place it on a cutting mat keeping the features on top.
Be careful not to crack the silicon wafer during this procedure.
9. Cover the microstructures with Scotch Magic tape and cut the
edges of the device cleanly, using a flat knife and the
24 Â 50 cm contour as a guide (see Note 20) (Fig. 1b).
10. To generate the two inlet and the two outlet ports, using the
biopsy punch, first pierce the device from the surface of the
1-mm circles until reaching the middle of the device, and then
pierce laterally the device rejoining the vertical hole perpendicularly (see Note 22).
Microfluidic Microscopy of Mycobacteria
215
by soaking the wafer into 10 mL of PGMEA twice for 10 min.
12. Wash extensively the wafer with isopropyl alcohol, using a
venting wash bottle, until all the whitish deposits disappear.
13. To make the microstructures more compact and robust, hard
bake the wafer on a hot plate at 180
C for 2 h.
14. Transfer the processed silicon wafer inside a vacuum chamber,
and dispense 200 μL of PFOCTS on a glass slide next to the
wafer. Apply the vacuum overnight to deposit a thin layer of
silanizing agent onto the surface of the wafer.
3.6 Soft Lithography
and Microfabrication
1. Secure the SU8-micropatterned silicon wafer to the bottom of
a square petri dish using fast-drying glue (see Note 21).
2. Pour 120 g of Sylgard 184 prepolymer into a plastic goblet and
add 12 g of Sylgard 184 curing agent base, for making the
PDMS mix.
3. Use a disposable L-shaped spreader to mix the reagents to a
milky consistency until the curing agent is evenly distributed
and the mixture appears full of air bubbles.
4. Place the goblet in the vacuum chamber and apply vacuum. As
the vacuum builds up, bubbles will surface. Vent the chamber a
few times initially to burst the bubbles and then leave it for
complete degassing for 15–30 min.
5. Pour the degassed PDMS mix onto the micropatterned silicon
wafer contained in the petri dish.
6. Place the petri dish in the vacuum chamber and apply vacuum.
As the vacuum builds up, a few bubbles will surface. Vent the
chamber a few times initially to burst the bubbles and then
leave it for complete degassing for 15–30 min.
7. Remove the petri dish and incubate in an oven at 80
C overnight to crosslink the PDMS.
8. Using a scalpel cut gently the PDMS block along the wafer
perimeter, peel off the PDMS block corresponding to the wafer
area, and place it on a cutting mat keeping the features on top.
Be careful not to crack the silicon wafer during this procedure.
9. Cover the microstructures with Scotch Magic tape and cut the
edges of the device cleanly, using a flat knife and the
24 Â 50 cm contour as a guide (see Note 20) (Fig. 1b).
10. To generate the two inlet and the two outlet ports, using the
biopsy punch, first pierce the device from the surface of the
1-mm circles until reaching the middle of the device, and then
pierce laterally the device rejoining the vertical hole perpendicularly (see Note 22).
Microfluidic Microscopy of Mycobacteria
215