The overall goal of the following experiment is to develop a low cost sensitive and rapid diagnostic for monitoring the health of barrier tissue. This is achieved by fabricating an organic electrochemical transistor, which can rapidly and sensitively monitor changes in ion flow in an electrolyte by measuring changes in the source drain current of the transistor. As a second step, integrate a healthy barrier tissue layer grown on a filter onto the OECT device to allow the monitoring of ion flow across the barrier tissue layer, which will be partially blocked if the cell layer is healthy.
If a toxic compound is added that destroys or disrupts the barrier tissue layer ion flow across the cell layer will no longer be blocked. Results are obtained that show the OECT can successfully detect the toxic effects of a model compound, demonstrating that we can use the measurements from the transistor as a diagnostic for monitoring the health of barrier tissue. The main advantage of this technique over existing methods such as impedance spectroscopy, is that organic electronic devices have the potential to be produced at low cost and are therefore amenable to high-throughput screening.
Demonstrating the procedure will be SHA FIA, a graduate student and Mark Hamir a postdoc in my laboratory. This method can provide insight into the cells of the gastrointestinal epithelium, but it can also be applied to other system such as the skin, the kidney epithelium, and the broadband barrier. Visual demonstration of this method is critical as a step needed to integrate a cell layer with a device.
Require careful manipulation To define thermally evaporated gold source and drain contacts via liftoff lithography. First spinco photo resist on a normal clean three inch by one inch glass. Slide at 3000 RPM for 30 seconds and bake the samples for 30 seconds at 110 degrees Celsius.
Next, define the patterns by photolithography with the use of a mask dedicated to the gold contacts inside an evaporating chamber. Deposit five nanometers and 100 nanometers of chromium and gold respectively. After that, lift off the photo.
Resist in an acetone bath for one hour, leaving the substrate with the source and drain gold contacts area only the the desired length of the pdot PSS channel is one millimeter, which is achieved by patterning using a Lene C peel off technique. Now load 3.5 grams of Lene C in the coating setup. Uniformly evaporate two microns of paraline C on top of the substrate with gold contact to pattern the channel by photolithography spin coat photo resist at 3000 RPM for 30 seconds and bake the substrate for two minutes at 110 degrees Celsius.
Next, define the patterns with the use of a mask dedicated to the channel area. The part of the photo resist, which is UV exposed, will become soluble in the developer afterward. Etch the Lene C in the channel area by exposing it to oxygen plasma for 15 minutes in the chamber in order to open the channel and the gold contact.
Then deposit the Pdot PSS mixture solution by spin coating at 500 RPM for 45 seconds and bake for 30 seconds at 110 degrees Celsius. Subsequently, peel off the Paraline C to reveal the Pdot PSS channel of the substrate underneath. This channel should slightly overlap with the gold contact.
Bake the samples for one hour at 140 degrees Celsius under atmospheric conditions. In this procedure, make PDMS by mixing the curing solution and the base solution at a ratio of one to 10 and bake for one hour at 120 degrees Celsius. Next, design a well using a hole punch and cut a square around the desired area.
Then glue A-P-D-M-S well on top of the channel to give a channel area of six square millimeters. Make a plastic support with a hole in it, then glue it on top of the PDMS and let it dry overnight. Next, test the well for leakage by pre-filling with water.
Begin this procedure by first preparing the cell culture media. Next, sterilize the cell culture media using a sterile filter device. Then maintain caco two cells in the cell culture media between passage 49 and 68 at 37 degrees Celsius in a humidified atmosphere of 5%CO2.
Divide the cells once a week using trypsin and seed at 1.5 times 10 to the fourth cells per insert. At the same time, change the cell culture media twice a week over three weeks. Now connect a silver, silver chloride wire to a source meter for use as a gate electrode.
Next, connect the source and drain to the source meter. Then immerse the tip in cell culture media, which is used as the electrolyte. Apply a square pulse positive voltage between the gate and the source.
Then apply a negative constant voltage between the drain and the source and measure the corresponding current. Enter the OECT parameters in a customized acquisition program. Read out the source drain current and gait current and carry out the measurement for several minutes to ensure a stable baseline signal for cell integration during off time of OECT measurement.
Remove the gait electrode from the electrolyte, then incorporate the cell culture insert and replace the gait electrode inside the cell culture insert. Next, carry out a baseline measurement with the cells for several minutes to ensure a stable signal. This baseline will be used as for calculation of the normalized value corresponding to an intact monolayer.
In this procedure, add EGTA solution in the appropriate volume to attain the desired concentration in the basal chamber during the off time, carry out the measurement continuously as previously described for 90 minutes. If the measurement is being carried out at room temperature, the stability of the cells will not remain constant after 90 minutes at the end of the run, scratch the cell layer to cause a complete destruction of the barrier layer and measure for 15 minutes. This baseline will be used for the calculation of the normalized value corresponding to a destroyed monolayer.
Here is the overview of the INCI two measurement of drain current response to the square gate pulses over time. This section corresponds to the OECT operated before the integration with ensuring the stability of the device. This section corresponds to the integration of the barrier tissue forming cells, again, ensuring a stable signal before commencing testing of toxic compound.
This section corresponds to the addition of EGTA to the barrier tissue cells, which is the evolution of the signal over time. Shown here is the OECT drain current response to a single square gait pulse. This figure shows the OECT transient response before and after the addition of barrier tissue forming cells grown on a filter and this figure shows the OECT transient response after the addition of toxic compound on a barrier tissue and without barrier tissue.
This graph shows the typical normalized results from the OECT. The normalized responses of the OECT were measured over a period of 50 minutes on the introduction of device indicated by an empty circle cell layer indicated by a black cross five millimolar EGTA, indicated by a dark blue circle and 10 millimolar EGTA indicated by a cyan circle. While attempting this procedure, it's important to remember not to image a cell layer with a gate electro or by pipetting the solution to strongly inside the cell culture insert.
Don't forget that working with live cells requires appropriate protective equipment and that articles that come into contact with life cells material should be disposed as a bio waste. After watching this video, you should have a good understanding of how to integrate a barrier tissue layer with the OECT for monitoring toxicology.
