To begin, cut a length of PTFE tubing for every control channel of the microfluidic device. Insert the pin of a 23-gauge half-inch lure stub at one end. Attach the lure stub to a male lure.
Then, insert the connector into a length of polyurethane tubing. Connect the other end of the polyurethane tubing directly to a solenoid valve. Next, connect a 23-gauge, 0.5 inch lure stub at the end of a syringe.
Fill the syringe with water. Attach the free end of the PTFE tubing to the syringe. Inject water until approximately halfway through the tubing.
Now, disconnect the tubing from the syringe and insert the free end into a punched hole of the corresponding control channel. Repeat until each control channel is connected to the corresponding solenoid valve. Next, launch the main interface program.
Press the Pressurize All Control Channels option to open the valves. This will push the fluid from the tubing into the control channels of the microfluidic device to fill it up. For each of the aqueous reagents, cut a segment of PTFE tubing long enough to connect the pumps to the microfluidic device inlets.
Connect a 23-gauge, 0.5 inch lure stub to the end of a syringe. Fill a syringe with the required reagent. Inject the reagent into the tubing until the tubing is full.
Insert the free end of the tubing into a corresponding inlet in the microfluidic chip. With the software, apply a pressure of 400 millibars to each of the inlet aqueous reagents. Sequentially, depressurize the control channels individually using the main interface program.
Press the corresponding icons on the program in the box labeled Control Channels Manual Pressurization to actuate individual valves if necessary. After repeating the pressurization for the oil reagents, click on Depressurize All Control Channels to simultaneously depressurize the channels. Now, click on Pressurize All Control Channels to repressurize the control channels.
And code the composition, sequence, and replicates of each plug population in a CSV file by marking the necessary control channels with a zero, if it's corresponding inlet needs to be open, or with a one if it needs to be closed. This will serve as input for the automatic experiment in the main interface program. Next, click on the folder icon in the Experiment File tab to load the CSV file.
Input the relevant fields such as Iterations of Experiment, Time of Depressurization, and Time of Pressurization. Then, choose the inlet channels corresponding to barcode production in the Barcode Inlets section, along with the duration for which they need to be open. Alternatively, the barcodes can be hard coded into the input CSV file.
Now, reduce the pressure of the inlet oil reagents from 400 millibar to 200 millibar. Next, connect a PTFE tubing to the outlet to collect the plugs. Use prefilled tubing to neutralize the difference in pressure at the outlet.
Lastly, press Run Experiment to start the program and plug production. The valves were able to regulate fluid flow until approximately 800 millibar of input pressure. At 1200 millibars, the input pressure was too high for flow regulation.
The flow rate of distilled water when injected at constant pressure dropped to zero. Upon depressurization, the flow rate recovered to the pre-actuation levels. However, under constant flow rate, the valve actuation did not result in complete inlet closure.
To demonstrate the device functionality, a quantitative combinatorial library of plugs was generated.
