7. Apply 35 bar pressure for 10 min after the temperature is

settled.

8. Leave the system for cooling to 60
C, under pressure for 1.5 h.

3.5

Thermo-

Compression Bonding

The last step of the microfluidic device fabrication is thermo-

compression bonding. A cover COP substrate with or without

electrodes are bonded to the COP substrate with microchannel

grooves and drilled inlet and outlet holes.

1. Set hydraulic press machine to 125
C.

2. Clean two glass slides, hot-embossed-COP substrate and

another COP substrate (with or without integrated electrodes)

similar to previous steps (see Note 8).

3. Align hot-embossed-COP substrate and the other COP sub-

strates. Stack these substrates between the two glass slides.

4. Wrap around the stack with aluminum foil and place in the

hydraulic press machine.

5. Apply warming up process for 10 min without pressure.

6. Apply 25 bar pressure for 45 min to proceed the bonding step.

7. Set the temperature controller of the hydraulic press machine

to 10
C for cooling under pressure.

8. Cooling process takes 3 h. Separate glass pieces from the

bonded substrates carefully without damaging.

9. Device fabrication is successfully completed (Fig. 2b, c) (see

Note 9).

3.6

Establishing the

Experiment

3.6.1

Microfluidic Device

Without Electrodes

1. Prepare all the necessary media (cell medium and nutrient

medium) and transfer them into separate syringes (5 mL).

2. Power on the syringe pumps and place the syringes on them.

3. Power on the inverted fluorescence microscope.

4. Place the fabricated microfluidic device on the stage of inverted

fluorescence microscope.

5. Connect the syringe pumps to the microfluidic device via

tubings.

6. Prime the microfluidic device with relevant medium (see Note

10).

7. Send cells through the middle inlet of the chip.

8. When chambers are filled with cells, stop the cell feeding and

start to send fresh medium through the side inlets for 3 h.

9. Feed medicated medium for another 3 h (see Note 11).

10. Stop medicated medium and send fresh medium for 10 h.

Fabrication Protocol for Thermoplastic Microfluidic Devices: Nanoliter. . .

35