The overall goal of this procedure is to describe the manufacturer of a custom made microdialysis probe. This is accomplished by first cutting polytetrafluoroethylene tubing and fused silica into the correct dimensions. The second step is to insert the fused silica into the tubing using a cannula as guidance.
Next, a dialysis membrane is positioned over the fused silica. The final step is to pull the metal sleeve onto the remaining part of the fused silica and to use epoxy to close off joints and to ensure an exchange length of two millimeters. Ultimately, the instructions in this video will show how to build a sturdy and reliable custom-made microdialysis probe as an alternative to a commercial probe, which can accurately measure in vitro recovery of neuropeptides and small molecules.
The main advantage of this technique is that this microdialysis probe assembly presented in detail can be adapted to any application and can be manufactured for less than 10. To begin, cut a piece of 1.6 millimeter thick polytetrafluoroethylene tubing with a 350 micron inner diameter into 2.5 centimeter long sections. Cut one end of the section at a 45 degree angle in order to make it easier to connect the outlet tubing.
Next, use sandpaper to roughen the outer surface of the PTFE tubing to promote adhesion of an epoxy glue. Cut a section of fused silica into a 2.25 centimeter long piece. Insert a 30 gauge needle into the hollow PTFE fiber one centimeter from the flat end.
Bend the PTFE and pierce the sheath at the bend. Insert the fused silica into the PTFE tubing using the needle cannula as guidance. Then gently remove the needle cannula and position the fused silica so that five millimeters project out the flat end of the PTFE tubing.
Use a drop of cyanoacrylate glue to secure the fused silica in place at the puncture site and allow it to harden overnight. Prepare the epoxy glue by adding 30 parts of yellow hardener to 70 parts of black epoxy glue and mixing gently. Then pull a 30 kilodalton cutoff polysulfone dialysis membrane with an inner diameter of 210 microns and an outer diameter of 245 microns.
Carefully over the fused silica. Pull the membrane into the PTFE tubing and adjust the membrane to 5.5 millimeters using a sharp scalpel. Mark a point on the membrane with a fine pen to define the exchange length of two millimeters from the end of the membrane.
Then use a fine needle to apply a small amount of epoxy glue to cover the tip of the membrane. Next, use epoxy glue to close off the joint between the dialysis membrane and the PTFE fiber. Now go back and add enough epoxy glue onto the membrane up to the marking point.
To ensure a diffusion exchange length of only two millimeters, allow the epoxy glue to harden overnight. Use a pair of pliers to carefully break off one centimeter pieces of a 25 gauge needle in order to create metal sleeves. Then pull the metal sleeve onto the remaining part of the fused silica and use the remaining epoxy glue to seal the joint between the metal sleeve and the PTFE tubing.
The metal sleeve later enables the connection of the inlet tubing with a microdialysis probe. Finally, apply hot glue around the bifurcation of the probe for additional fixation and stabilization of the device. This schematic shows a closeup of the inner workings of the microdialysis probe.
Two millimeters of the polysulfone dialysis membrane is exposed at the end of the probe, which allows for the recovery of small molecular weight metabolites such as glucose a TP, and acetylcholine. The ability of the probe to recover small, low molecular weight molecules such as glucose, lactate, acetylcholine, and a TP in vitro is shown here. The percent recovery is much higher with these molecules, which are around 500 Daltons than larger molecules such as angiotensin two, substance p, and somatostatin, which are above 1000 Daltons.
After watching this video, you should have a good understanding how to build sturdy and reliable microdialysis probes as an alternative to commercial ones.
