Fabrication of PDMS-based Acoustouidic Devices for 3D Access to Single Cells and Small Organisms

Acoustouidic manipulation has demonstrated signicant potential for the investigation and handling of small organisms. In this protocol, we describe the steps required for the fabrication of a PDMS-based device with 150 um high structures to be used for the rotational manipulation of single cells and small organisms.

-Clean the wafer with acetone in an US bath for 5 minutes.
-Clean the wafer with IPA in an US bath for 5 minutes -Remove the solvents with de-ionized water using a QDR until a resistivity of 12 MOhm cm is reached.
-Dry the wafer in a spin-rinse-dryer.
-Bake the wafer at 200 °C on a preheated hotplate for 5 minutes.
-Remove the wafer from hotplate and let it cool down to room temperature. -Bake the coated wafer at 65 °C on a preheated hotplate for 5 minutes.
-Increase the temperature of the hotplate to 95 °C and bake the wafer for additional 30 minutes.
-After a total of 35 minutes, turn off the hotplate and let the wafer cool down to 75 °C while still on the hotplate.
-Remove the wafer from the hotplate and let it cool down to room temperature. Exposure: -Measure the energy density (mW/cm2) of the UV lamp at a wavelength of 365 nm.
-Load the mask and the wafer into the mask aligner and align them.
-Expose the photoresist until an exposure dose of 260 mJ/cm2 is reached.
Post-exposure bake: -Bake the exposed wafer at 65 °C on a preheated hotplate for 5 minutes.
-Increase the temperature of the hotplate to 95 °C and back the wafer for additional 12 minutes.
-After a total of 17 minutes, remove the wafer from the hotplate and let it cool down to room temperature. -Move the wafer to a second Petri dish with fresh developer for additional 30 seconds.
Rinse, dry and hardbake: -Rinse the wafer and the developed SU-8 structures with IPA.
-Rinse the wafer with de-ionized water.
-Dry the wafer with a nitrogen gun.
-Hardbake the wafer at 150 °C on a preheated hotplate for 5 minutes.
-Remove the wafer from the hotplate and let it cool down to room temperature.
Silane coating -Plate the wafer with the SU-8 structures next to a glass slide in a vacuum chamber.
-Add three drops (each about 50 uL) of silane on the glass slide.
-Close the chamber and start the vacuum pump.
-Once su ciently low pressure (less than 100 mbar) is reached, turn off the vacuum pump and let silane distribute inside the chamber for 30 minutes.
-Remove the wafer from the vacuum chamber and bake it on a hotplate at 120 °C for 10 minutes.
-Remove the wafer from the hotplate and let it cool down to room temperature.
PDMS casting -Mix curing agent and pre-polymer with a weight ratio of 1:10 in a disposable cup for 5 minutes.
-Place the wafer in a Petri dish inside a vacuum chamber.
-Cover the wafer with the PDMS and apply a vacuum for 30 minutes to degas the polymer and ensure proper molding of the SU-8 structures. Bleed the vacuum occasionally to accelerate the process and to prevent PDMS from over owing the Petri dish.
-Cure the PDMS by placing the Petri dish containing the mold and the degassed polymer in an oven and bake it for 1 hour at 80°C.
-Remove the Petri dish with the cured PDMS from the oven and let it cool down to room temperature.
Final steps -Cut the PDMS and peel it off the silicon wafer. If necessary, punch the connectors using a biopsy punch. For 3D access to the manipulated specimen (see associated publication), cut the fabricated PDMS channels perpendicularly.
-Place the PDMS (microchannels facing upwards) next to a glass slide in a plasma asher.
-Expose the glass slide and the PDMS to oxygen plasma for 30 seconds.
-Bring the exposed surfaces in contact to chemically seal the structures.
-Fix a piezoelectric transducer used for subsequent acoustic excitation on the glass slide near the PDMS structure using a suitable glue, e.g., two-component epoxy.

Troubleshooting
White clouds during IPA rinse after development: Clean the wafer with water and dry it. Repeat the second development step.