The overall goal of this procedure is to identify abnormalities in neuronal and neuromuscular signaling in the C Allergan's model organism following chemical exposure or genetic manipulation. This is accomplished by first designing and etching a microchannel pattern into a silicon wafer. During the second step, the silicon mold is used to cast one or more micro channels from PDMS.
Then accessory components are attached to the PDMS mold. Next, the behavior of the worms as they are stimulated to swim through the microchannel is recorded using a microscope mounted camera. Ultimately, electro microfluidics is used to determine the changes in the nematodes, neuronal, and muscular systems due to chemical or genetic manipulation with locomotion as the readout.
So the main advantage of this technique over the existing techniques, especially the plate based behavioral assays, is that with this technique, the direction of locomotion of the worm can be tightly controlled, allowing for precise quantification of the locomotive behaviors such as body bend frequency, rotation time, and swimming speed. The implications of this technique extend toward the therapy of neurodegenerative disorders such as Parkinson's and Alzheimer's because it can be used to screen for chemical and genetic factors with the neuroprotective properties. We first had the idea for this technique when we realized that there is lack of method for analyzing the method movement behavior in a quantitative manner.
Movement is voluntary and random, and it's hard to characterize. So we wanted to establish a easy method that everyone can learn To make the master mold begin by bathing a three inch silicon wafer in acetone and then methanol for 30 seconds each. Then rinse the wafer with deionized water for five minutes.
Next, use a nitrogen blow gun to dry the wafers surface and then heat the wafer on a hot plate at 120 degrees Celsius after two minutes, plasma oxidize the surface of the silicon wafer at 50 watts for one minute. Now using three milliliters of SU eight 100 photo resist spin coat the wafer surface at 1, 750 RPM for 40 seconds, and then pre-bake the coated wafer on a hot plate at 65 degrees Celsius. After 10 minutes, increase the temperature to 95 degrees Celsius over two minutes and bake the wafer for an additional hour.
After removing the wafer from the hot plate, arrange a photo mask of the desired design over the wafer. Expose the photo resist to UV light for 95 seconds. Then post bake the wafer on a hot plate using the same temperature gradient as used for the pre-baked after post baking.
Immerse the wafer in SU eight. Develop a solution for 10 to 15 minutes. Rinse the wafer with isopropanol to confirm the completion of the design development.
Then rinse with deionized water for 30 seconds. Finally, dry the wafer with a nitrogen gun and bake briefly at 120 degrees Celsius. Now, place the fabricated master mold as well as a blank silicone wafer into two separate Petri dishes lined with aluminum foil.
Pour 20 milliliters of PDMS pre polymer into the master mold and dish, and 15 milliliters into the blank wafer dish. Next, gently press on the wafers with a disposable wooden applicator to eliminate any air pockets underneath the wafers, and then cover both dishes and set them aside for a day to cure. After the wafers have cured, remove the foil and peel away the PDMS.
Then use a 2.5 millimeter Harris unicorn to punch fluid access ports at both ends of the channel in the PDMS from the master mold. Cut both PDMS discs into similarly sized strips. Then in a clean room, load the channel, the blank PDMS strip and a glass slide into a plasma oxidizer.
Expose the materials to oxygen plasma at 40 watts of power for 40 seconds. Then stick the channel piece and the glass slide to opposite sides of the blank strip and set the materials aside to complete the bonding. After two hours, use PDMS pre polymer to attach two pieces of plastic tubing at least six inches long to the punched reservoirs on a hot plate at 120 degrees Celsius.
Allow the PDMS to cure before continuing. Then affix a fluidic plastic connector to one of the tubes to allow syringe attachments. Now, insert three inch lengths of 22 gauge insulated copper wire into each reservoir between the inlet tube and the channel securing the wires with more PDMS pre polymer.
Begin this step by placing the just assembled microchannel onto an XY movable stage of a microscope with a mounted camera connected to a monitor, connect the power supply to the microchannels electrodes. After confirming that the resistance of the microchannel is around 0.6 mega ohms, attach the output tube of the microchannel to a disposable syringe. Then submerge the mouth of the inlet tube into a solution of nematodes suspended in M nine physiological buffer.
Apply negative pressure inside the syringe to aspirate liquid into the channel. When the inlet and outlet tubes are both filled, disconnect the syringe and hydrostatic. Manipulate the flow by adjusting the tubes relative height to place a worm in the center of the channel.
Then lay both tubes flat at the same elevation to maintain zero flow inside the microchannel. When switching worm samples during an electro taxis experiment, after leveling both the inlet tubes to the same elevation, if there is still flow, make small adjustments to the tube's height relative to each other. If we are ever unsure of whether the flow is really zero, we can induce a reversal in the worm's movement by changing the polarity of the electric field and then evaluating the worm's linear velocity as it turns around.
Now set the power supply to the appropriate voltage. Activate the electric signal and allow one minute of pre-exposure for the worm to acclimatize to the field during which time the worm should begin moving towards the cathode when the minute has passed. Begin recording.
When the experiment is finished, remove all the liquid and worms from the channel. Rinse the chamber with deionized water and leave the device on a hot plate at 125 degrees Celsius to dry. In this representative video, a wild type young adult nematodes electro axis and its position and velocity outputs from the worm tracking software are shown.
The video begins with the cathode on the right. The appearance of the glass pipette indicates the impending reversal, subsequent removal of the pipette signals. The moment of reversal here, the data for position versus time and instantaneous velocity versus time outputs of the custom worm tracking program for the worm In the video are shown the program calculated the velocity curve from the position time curve.
This box plot displays electro speed data from a set of wild type and trenchgenic animals. The T transgenic animals express the human alpha-synuclein gene in the body wall muscles under the control of the UNC 54 myosin heavy chain gene promoter, which causes protein aggregation and manifests as a slower electro tactic response than that of wild type animals. Once mastered, more than 20 worms can be assay every hour if the electro taxis experiment is performed properly.
While attempting this procedure, it's important to remember that only mutations and chemicals affecting locomotion or electros sensation will produce phenotypes that are detectable by the electro taxes assay. After watching this video, you should have a good understanding on how to pattern a silicone. We assembled microfluidic channel and how to analyze locomotion of N method using an axxis assay.
