Target Peptide Detection and Measurement Using a Capacitive Immunoprobe Biosensor

Protokoll

1. Capacitive immunoprobe fabrication and functionalization

1. Cut a 25 cm length of perfluoroalkoxy (PFA)-coated platinum wire (see Table of Materials) and strip approximately 5 mm of PFA coating from one end using a scalpel, being careful not to cut into the platinum wire.
2. Insert the stripped end of the platinum wire into a gold-plated 1 mm male connector pin and crimp the connector pin teeth around the stripped end of the platinum wire using needle-nose pliers (see Table of Materials).
3. Solder the platinum wire to the gold-plated connector pin. Be careful not to use an excessive amount of solder.
4. Prepare dopamine solution by dissolving 50 mg of dopamine HCl in 50 mL of 10 mM phosphate-buffered saline (PBS, pH 6.0) by stirring.
5. Once the dopamine is completely dissolved, place the tip of the platinum wire in the vessel containing the freshly made dopamine-supplemented PBS. Plug a gold connector pin into a channel of the headstage (see Table of Materials).
6. Connect the silver chloride (AgCl) disc electrode (ground electrode, see Table of Materials) to the ground channel in the headstage. Place the AgCl disc in the vessel containing dopamine-supplemented PBS and platinum wire; be careful only to submerge the disc electrode and not any length of the wire or solder. Connect a wire shunt into the reference channels of the headstage prior to proceeding.
7. Open the interactive data acquisition software (see Table of Materials). Prepare a sawtooth electrodeposition command potential protocol with the following parameters: start potential = −0.6 V; end potential = +0.65 V; scan rate = 0.04 V∙s^-1; duration of deposition = 420 s. Begin the polydopamine deposition protocol, ensuring that all the wires are connected properly.
8. After completing the polydopamine deposition, remove the AgCl ground pellet and the tip of the platinum wire from the vessel, being careful not to disturb the tip of the platinum wire electrode. Place the tip of the wire into a microtube containing PBS (pH 7.4) for 2-5 min as the antibody solution is prepared; ensure the wire tip does not contact the sides or bottom of the microtube.  
   NOTE: Antibody solution can be made during polydopamine deposition; however, the transfer of the platinum wire from the dopamine-containing vessel to the microtube of PBS following polydopamine deposition should not be skipped.
9. Prepare the antibody solution. Combine the antibody of interest with PBS (pH 7.4) in a 1:20 ratio in a vessel of an appropriate size (e.g., a microtube).
   NOTE: The anti-neuropeptide Y (anti-NPY) monoclonal antibody (see Table of Materials) used here was aliquoted at 1 mg/mL; an example antibody preparation here would be 4 µL of antibody to 76 µL of PBS.
10. Soak the polydopamine-deposited tip of the platinum electrode in antibody solution for a minimum of 2 h at room temperature, again ensuring the platinum wire tip is suspended in solution and not resting on the interior surface of the microtube.
    NOTE: Recent implementation of this technique has favored using the platinum wire electrode immediately following this step instead of wet or dry storage for later use.
11. After soaking in the antibody solution, briefly rinse the newly-functionalized capacitive immunoprobe (CI probe) tip in PBS (pH 7.4). The probe is now ready for use.

2. Experimental setup for in vitro detection and measurement of peptide

1. Place the functional tip of the CI probe 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.  
   NOTE: The flow chamber was created by pouring silicone elastomer (see Table of Materials) into a 35 mm culture dish with an elongated ovoid space-filler in the center of the dish. After hardening, the ovoid shape is removed from the elastomer. The chamber is then superfused with Tris-buffered saline (TBS) and allowed a flow rate of 3 mL/min. Ensure the inflow and outflow maintain the fluid level in the chamber such that no tidal action of the superfusate is observed. The flow must remain in place for as long as the CI probe is in use.
2. Prior to the first experimental test, perform a TBS standard run to condition the CI probe. Set up the following command voltage protocol: positive step potential = +100 mV; negative step potential = −5 mV; step duration = 20 ms; duration of acquisition = 600 s.       
   NOTE: It is important to allow for the equilibration of the probe during the initial phase of cycling command potential prior to data acquisition.
3. Create a solution of the peptide 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 without introducing bubbles into the tubing system or flow chamber.  
   NOTE: Synthetic porcine NPY peptide (see Table of Materials) was used in the present study.
4. Set up the peptide-sensing data acquisition protocol using TBS standard parameters (see step 2.2.).  
   ​NOTE: In this implementation, the duration of each experimental test was 360 s (120 s TBS, 120 s peptide-supplemented TBS, 120 s TBS).

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Materialien

Name	Company	Catalog Number	Comments
AgCl disc electrode	Warner Instruments (Holliston, MA)	64-1307
Anti-NPY monoclonal antibody	Abcam, (Cambridge, MA)	ab112473
Custom multichannel amplifier/ 1 MΩ feedback resistor multichannel headstage	NPI Electronic, (Tamm, Germany)	NA	Based on NPI VA-10M multichannel amplifier
Dopamine HCl	Sigma Aldrich (St. Louis, MO)	H8502-10G
Gold-plated male connector pin	AMP-TE Connectivity (Amplimite)	6-66506-1
HEKA LIH 8+8 analog-to-digital/digital-to-analog device	HEKA Elektronik, (Holliston, MA)	NA
Igor Pro data acquisition software, v. 7.08	WaveMetrics, (Lake Oswego, OR)		Software driving command potential and data acquisition was custom written
Masterflex L/S Standard Digital peristaltic pump	Cole Palmer, (Vernon Hills, IL)
PFA-coated platinum wire	A-M Systems, (Sequim, WA)	773000	0.005" bare diameter, 0.008" coated diameter
Silicone elastomer	World Precision Instruments (Sarasota, FL)	SYLG184
Synthetic porcine NPY peptide	Bachem (Torrance, CA)	4011654