This is the first time a method has been developed for the time-resolved measurement of peptide transmitter levels in vivo. We can measure peptide transmitter levels in single subjects and discrete locations with near-realtime analysis. First, cut a 25-centimeter length of perfluoroalkoxy-coated platinum wire.
Then, using a scalpel, strip approximately five 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, one-millimeter male connector pin. Then, using needle-nose 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 six 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 disc electrode to the ground channel in the head stage.
Then, place the silver chloride disc 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 electro-deposition 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 poly-dopamine 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 two to five 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 1:20 ratio in an appropriate size vessel.
Next, soak the poly-dopamine 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 probe, and for the voltage protocol, set the positive step potential to 110 millivolts, negative step potential to minus five 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.
Before the poly-dopamine deposition, thread the exposed tip of the platinum wire electrode through a 22-gauge hypodermic needle, leaving approximately two millimeters beyond the tip of the needle. Use forceps and gently bend the tip of the platinum wire electrode to create a barb that hangs from the end of the hypodermic needle. Gently withdraw the needle from the barbed tip, leaving enough wire to place in the vessel without the needle contacting the fluid and proceed with dopamine deposition as demonstrated previously.
Now, rinse the barbed probe tip with PBS and place it in the antibody solution. Then, gently remove the functionalized tip from the PBS. Move the hypodermic needle to the barbed capacitive immunoprobe and gently implant it in the region-of-interest before plugging the gold connector pin into the head stage.
Once implanted, withdraw the hypodermic needle, leaving the electrode in place. A positive step in the command potential measures the injection current required to clamp the probe voltage and allows the measurement of the capacitive current. The negative command potential step clears the bound peptide from the antibody via electrostatic repulsion.
The step function command waveform depicted the time-resolved iterative detection and quantification of the peptide. The bottom trace represented the command potential and the resulting current under the superfusion of the probe in TBS. The equilibrated capacitive immunoprobe showed smaller initial decay and a stable baseline over six minutes.
The flow of neuropeptide Y into the flow chamber increased the capacitive currents over baseline. Capacitive immunoprobe currents measured with a neuropeptide Y antibody-functionalized probe showed a concentration-dependent signal sensitive to low-picomole neuropeptide Y For calibration, a standard curve was measured for all tested concentrations. The stimulation of the bilateral stellate ganglion showed elevated neuropeptide Y when co-plotted with the negative control capacitive currents.
Norepinephrine release measured under stellate stimulation showed a synchronous release with neuropeptide Y, consistent with an evoked sympathetic response. The development of these techniques may provide intraoperative readouts of key modulators of cardiac activity, thus providing potential rapid therapeutic or interventional guidance. The technique as presented here is specific for cardiovascular deployment, however, the technique itself is appropriate for other physiological settings, such as urine, skeletal muscle, kidneys, adipose tissue, et cetera.
