Target Peptide Detection and Measurement Using a Capacitive Immunoprobe Biosensor

First, cut a 25-centimeter length of perfluoroalkoxy-coated platinum wire. Then, using a scalpel, strip approximately 5 millimeters of perfluoroalkoxy coating from one end, careful not to cut into the platinum wire. Now, insert the stripped end of the platinum wire into a gold-plated, 1-millimeter male connector pin. Then, using needlenose pliers, crimp the connector pin teeth around the stripped end of the platinum wire, and solder the platinum wire to the gold-plated connector pin.

Next, prepare the dopamine solution by adding 50 milligrams of dopamine hydrochloride in 50 milliliters of 10 millimolar PBS, with pH 6, and mix by stirring. After completely dissolving the dopamine, place the tip of the platinum wire in the vessel containing the freshly made dopamine-supplemented PBS. Connect the silver chloride disk electrode to the ground channel in the head stage. Then, place the silver chloride disk in the vessel containing dopamine-supplemented PBS and platinum wire.

Before proceeding further, connect a wire shunt to the reference channels of the head stage. Open the interactive data acquisition software, and set a sawtooth electrodeposition command potential protocol by setting the start potential at minus 0.6 volts, the end potential to 0.65 volts, the scan rate at 0.04 volts per second, and the duration of deposition to 420 seconds.

After completing the polydopamine deposition, remove the silver chloride ground pellet and the tip of the platinum wire from the vessel. Place the wire tip into a microtube containing PBS of pH 7.4 for 2 to 5 minutes and ensure the wire tip does not contact the sides or bottom of the microtube. Then, prepare the antibody solution by combining the antibody of interest with the PBS of pH 7.4, in a 1-to-20 ratio, in an appropriate-size vessel.

Next, soak the polydopamine-deposited tip of the platinum electrode in antibody solution for two hours at room temperature, ensuring that the platinum wire tip is suspended in solution and not resting on the interior surface of the microtube. Place the functional tip of the capacitive immunoprobe into the flow chamber, taking care not to disturb the tip of the electrode in any way, as doing so may damage the sensory tip of the probe.

Before the first experimental test, perform a TBS standard run to condition the capacitive immunoprobe, and for the voltage protocol, set the positive step potential to 110 millivolts, negative step potential to minus 5 millivolts, step duration of 20 milliseconds, and duration of acquisition to 600 seconds.

Next, create the peptide solution of interest, using the same TBS. To maintain the superfusate's composition, set up a manifold system where the superfusate can be switched between TBS and peptide-supplemented TBS. Use TBS standard parameters, and set up the peptide-sensing data acquisition protocol by setting the duration of acquisition to 360 seconds.