Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Summary

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.

Abstract

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Introduction

Several chemical methods for detecting and quantifying biomarkers are routinely utilized in both protein chemistry and clinical diagnostics, particularly in cancer diagnoses and the assessment of cardiovascular disease progression. Currently, methods such as high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), and mass spectrometry rely on sample collection from the vascular compartment^1,2,3 by bulk fluid draw or the interstitial compartment by microdialysis. Microdialysis employs a semipermeable membrane tube of known length that is placed in a region of interest. Collection fluid is perfused through the tube over several minutes⁴ to collect the sample for analysis⁵, thus limiting temporal resolution. In this manner, samples collected only provide an averaged value over time of the local microenvironment and are limited by the perfusion rate and collection of sufficient sample volume. Moreover, these methods require the pooling of experimental data and signal averaging; therefore, they may fail to account for variability between subjects. Importantly, the time between sample collection and subsequent offline analysis precludes immediate clinical intervention and therapeutics.

In the present protocol, the use of a capacitive immunoprobe biosensor (CI probe) for the time-resolved electrical detection of specific bioactive peptides is outlined. Neuropeptide Y (NPY), released from post-ganglionic sympathetic neurons that innervate the vasculature, endocardium, cardiomyocytes, and intracardiac ganglia, is a major neuromodulatory peptide transmitter in the cardiovascular system^6,7,8,9. The method presented here is designed to measure NPY, and the experimental feasibility is demonstrated in a porcine heart model. However, this approach applies to any bioactive peptide for which a selective antibody is available¹⁰. This method relies on the capacitive junction between a platinum wire probe and the conductive fluid at the functionalized tip^11,12. In this application, the interaction was mediated through an antibody against the target neuropeptide (NPY), which was bound to the electrode tip, interfacing the conductive fluid environment. This functionalization was achieved through electrodeposition of reactive polydopamine onto the tip of the platinum wire probe^10,13.

When the antibody-functionalized probe is placed in a region of interest in vivo, evoked endogenous NPY release leads to binding to the trapping antibodies on the probe tip, and the conductive fluid at the electrode surface is displaced by the NPY protein. Local alteration in the electrical environment results in the displacement of high-mobility, high-dielectric fluid with an immobile, statically charged molecule. This alters the electrode-fluid interface and, thus, its capacitance, which is measured as a change in charge current in response to a step-function command potential. A negative "reset" potential is employed immediately following each individual measurement cycle to repel bound NPY from the antibody through electrostatic interaction, thus clearing the antibody binding sites for subsequent rounds of measurement¹⁰. This effectively allows for the measurement of NPY in a time-resolved manner. The unique CI technique overcomes the limitations of the microdialysis-based immunochemical methods outlined above to measure dynamic biomarker levels from a single experiment without data pooling or signal averaging over several experiments⁹, providing data in nearly real time. Moreover, the ability to adapt this method to any biomarker of interest for which there exists an appropriate antibody on a time-resolved and localized scale provides a major technical advance in immunochemical measurement for the evaluation of disease progression and the guidance of therapeutic interventions.

The software for data acquisition and analysis was custom-written in IGOR Pro (a fully interactive software environment). An analog to digital converter (A/D) system issued a command voltage under computer control and acquired data from a custom amplifier. The amplifier possessed certain unique features. These included a feedback resistor (switchable) for each of the four acquisition channels, allowing for choosing 1 MOhm or 10 MOhm feedback voltage clamp circuits to integrate the variability of the electrode. A stage unit with a single head and mutual ground/reference circuit for all the four acquisition channels was also built to place the device close by the chest in a single physical module. A 1 MOhm feedback resistor setting was used to collect all the reported data.

The filter and gain settings were telegraphed from the amplifier and recorded within the data file. Data were filtered at 1 kHz via a 2-pole analog Bessel filter digitized at 10 kHz. The difference in potential between the probe and surrounding conductive solution creates a Helmholtz capacitive layer at the probe tip. Ligand binding to the antibody at the probe tip results in an altered local charge and, thus, a change in the Helmholtz capacitance. This change in the capacitive component of the circuit results in a shift in the magnitude of injected charge required to bring the probe to the potential in the step-function voltage protocol. Thus, the binding of a specific ligand to the functionalized probe results in an alteration in the electrode capacitance measurement as a change in peak capacitive current.

Protocol

All animal experiments were approved by the University of California, Los Angeles Animal Research Committee and performed following the guidelines set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th edition, 2011). Adult male Yorkshire pigs of approximately 75 kg were used for in vivo studies¹⁰.

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 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-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).

3. Adaptation of the CI probe for in vivo use

1. Prior to polydopamine deposition (step 1.7.), thread the exposed tip of the platinum wire electrode through a 22 G hypodermic needle, leaving approximately 2 mm beyond the tip of the needle. Using forceps, gently bend back the tip of the platinum wire electrode, creating a "barb" that hangs from the end of the hypodermic needle.
   1. Gently withdraw the needle from the barbed tip, leaving enough wire to place in the vessel without the needle contacting the fluid. Proceed with steps 1.4.-1.11.
      NOTE: Depending on the in vivo setup, it may be necessary to cut a length of platinum wire longer than 25 cm.
2. Before connecting the CI probe to the headstage, ensure the entire electrical setup is properly grounded. Failure to do so may introduce unwanted electrical interference during the experimental recordings.
3. Anesthetize the animals following a previously published report¹⁰.
4. Perform surgery to expose the region of interest.
   NOTE: A median sternotomy was performed in the present study to expose the heart. Please see Kluge et al.¹⁰ for details on animal surgery.
5. Gently remove the functionalized tip from the PBS rinse (step 1.11.), forward the hydodermic needle to the barbed C.I. probe and gently implant it in the region of interest prior to plugging the gold connector pin into the headstage. Once implanted, withdraw the hypodermic needle, leaving the electrode in place.
   NOTE: For the present study, the probe was placed in the mid-lateral wall of the left ventricular myocardium¹⁰.
6. After ensuring proper electrical setup, proceed with standard and experimental testing protocols (step 2.2. and step 2.4.).
7. Upon completion of the experiments, euthanize the animal following institutionally approved techniques.
   NOTE: In the present study, the animals are euthanized under deep anesthesia via induction of ventricular fibrillation¹⁰.

Representative Results

Electrode fabrication and characterization
A flexible capacitive immunoprobe (CI probes) was fabricated, and a representative image is depicted in Figure 1A. The electrode potential was set by a computer-controlled voltage clamp circuit (Figure 1B), and the electrode was immersed in a polydopamine solution made in PBS. Polydopamine was electrodeposited onto the conductive electrode tip¹³ for functionalization. The command potential drove the probe voltage, and the clamping current injection was measured. The measured clamping current during a single cycle of polydopamine deposition is shown in Figure 1C. Immediately following polydopamine deposition, the coated electrode tip was soaked in an antibody solution of interest (Figure 2A). All electrodes were used immediately following these steps.

In vitro measurement of NPY
The antibody-functionalized probe was placed in a flow chamber and driven by the voltage clamp circuit with an example command potential, and the recorded current is shown (Figure 2B,C). In vitro recordings were performed in Tris-buffered saline (TBS, pH 7.4). The step-function waveform comprises a positive step potential (upper blue portion, Figure 2B) and a negative "reset" potential (lower red portion, Figure 2B). The range of this command potential effectively measures the binding and evokes subsequent repulsion of peptides without degrading the probe sensitivity¹⁰. After fabrication, each probe was tested for initial conditioning under the command potential protocol. It is to be noted that the probes that returned the most stable and reproducible initial conditioning profile, defined as a smaller initial decay followed by a stable baseline (Figure 2D), gave the most reproducible measurements in vitro and in vivo. Thus, measuring the initial stabilization of the CI probes represents an important quality control point to determine probe suitability.

Assessment of electrode sensitivity and stability in vitro
Capacitive immunoprobes were placed in a flow chamber and superfused with TBS supplemented with known concentrations of neuropeptide Y (NPY, 5-150 pM). Capacitive currents, measured as the peak amplitude of the +100 mV depolarization, were plotted against time. The flow of NPY into the flow chamber increased the capacitive currents over baseline (60 pM NPY, Figure 2E). A demonstration of the dose-dependent current response for 5 pM and 75 pM NPY is shown in Figure 3A, and a standard curve measured under all tested concentrations is provided in Figure 3B. The data points in Figure 3B were fit to a single exponential fit for calibration purposes.

In vivo assessment of neuropeptide release profiles with CI probe
This methodology was applied to a porcine heart preparation to assess its suitability for biological application, as described in the previous publications¹⁰. Briefly, a CI electrode was threaded through a 22 G hypodermic needle and bent back slightly to create a barb (Figure 4A). The needle and barbed electrode were inserted into the left ventricular myocardium of a beating porcine heart. The hypodermic guide needle was then withdrawn from the electrode, leaving it in place in the myocardium. Interstitial NPY was measured before and in response to 60 s of bilateral stellate ganglion stimulation (see Kluge et al.¹⁰ for a full description of the simulation steps). A second negative control electrode with no antibody functionalization was placed adjacent to the NPY probe. The capacitive immunoprobe clamp currents obtained were fitted against the in vitro calibration curve to provide temporally resolved, evoked changes in concentrations of NPY in the cardiac microenvironment. Elevated NPY was observed during stimulation of the bilateral stellate ganglion and was co-plotted with the negative control C.I currents (Figure 4B, upper plot). In parallel, interstitial norepinephrine levels (NE) were also measured by fast scanning cyclic voltammetry (FSCV), a technique previously utilized to assess the release of catecholamine from isolated cells^14,15, isolated tissues^16,17,18, and in the porcine heart preparation¹⁹. Norepinephrine is a sympathetic neurotransmitter co-released under stellate stimulation (Figure 4B, lower plot) and shows a synchronous release with NPY, consistent with an evoked sympathetic response. These data demonstrate that the CI approach can be used concurrently with other biomarker detection methods to assess interstitial microenvironments with high temporal fidelity. Briefly, a sensor electrode is implanted in the myocardium adjacent to the CI probe. The electrode potential is driven through the oxidation/reduction potentials of the transmitter by a voltage clamp circuit. Therefore, as the electrode potential becomes positive to the oxidation potential for NE, the NE is then oxidized to a quinone derivative. Electrons are generated by the oxidation reaction, measured as a compensating current in the voltage-clamped electrode, thereby providing an index of local NE release. Switching the electrode potential back to a negative polarization diminishes the quinone product to regenerate the catecholamine²⁰.

Figure 1: Fabrication of the platinum wire CI probe. (A) A PFA-coated platinum wire (25 cm long and 127 µm in diameter) was used to create each CI probe. Around 5 mm of PFA coating was stripped from one end of the wire, which was then soldered to a male gold-plated connector pin (scale bar = 3 cm). (B) A depiction of the voltage clamp circuit used to apply the command potential is shown. (C, left) The sensory tip of the electrode was immersed in a tube containing PBS supplemented with 5 mM dopamine. (C, right) Polydopamine was electrodeposited onto the sensory tip. Electrodeposition was performed by the use of a sawtooth waveform with the following parameters: −0.6 V to +0.65 V at a scan rate of 0.04 V∙s^-1. The command potential (top) and the resulting current (bottom) are shown. Please click here to view a larger version of this figure.

Figure 2: In vitro measurement of NPY with the CI probe. (A) A polydopamine-functionalized wire was immersed in PBS supplemented with an anti-NPY antibody for 2 h. This allowed the antibody to bind covalently to the polydopamine surface at the probe tip. (B) The voltage command potential is shown. A positive step in the command potential serves to measure the injection current required to clamp the probe voltage (blue) and allows the measurement of the capacitive current. The negative command potential (reset potential) step clears the bound peptide from the antibody via electrostatic repulsion (red). (C) The step function command waveform (top trace; +100 mV measure phase to −5 mV reset phase, each of 20 ms duration) provides time-resolved, iterative detection and quantification of the peptide. The command potential and the resulting current recording under superfusion of the probe with TBS is shown (bottom trace). (D) The equilibration of a CI probe over a 6 min period is shown (see step 2.2.). (E) The peak capacitive current measured by the NPY-functionalized C.I. probe is shown. The CI probe was superfused first with Tris-buffered saline, then Tris-buffered saline supplemented with 60 pM NPY, and then followed by an NPY-free Tris-buffered saline wash. Please click here to view a larger version of this figure.

Figure 3: In vitro calibration of the CI probe. (A) Capacitive immunoprobe currents were measured with an NPY antibody-functionalized probe superfused with Tris-buffered saline. The superfusate was switched to contain 5 pM (red) and 75 pM (black) NPY. These recordings represent a concentration-dependent signal sensitive to low picomole NPY. (B) Capacitive immunoprobes functionalized with the NPY antibody were tested for dose-response sensitivity. Recordings were performed under superfusion with TBS; then TBS supplemented with NPY (in pM): 1, 5, 15, 25, 60, 75, and 150, and then in an NPY-free TBS wash. Peak capacitive current values (C.I. Current) were measured at a steady state in each NPY concentration and plotted against NPY concentration. The dose-response data were fitted to provide a standard curve for calibration of the experimental data (adapted from Kluge et al.¹⁰). Please click here to view a larger version of this figure.

Figure 4: Differential release of NPY and NE evoked by bilateral stellate stimulation. (A) A CI probe with a barbed end for in vivo use is shown (scale bar = 3 mm). The probe is threaded through a 22 G hypodermic needle and prepared as mentioned in step 1. and step 2. Before inserting the sensor, the probe tip is bent to create a barb, and the needle is withdrawn upon insertion into the myocardium, thereby leaving a barbed probe anchored in the myocardial wall of the left ventricle. (B) A probe functionalized with an anti-NPY antibody was inserted into the left ventricular myocardium. An identical probe without antibody functionalization (ø mAb) was inserted immediately adjacent and served as a negative control. CI currents were determined in response to bilateral stellate stimulation at 10 Hz (BSG, 4 ms pulse width, 2x threshold). The resulting currents were calibrated against the standard curve (Figure 3B) and plotted (upper plot). Norepinephrine release in the same interstitial location was measured using fast-scanning cyclic voltammetry (lower plot). Please click here to view a larger version of this figure.

Discussion

The present protocol describes the manufacture and testing of a capacitive immunoprobe (CI probe) capable of detecting and measuring biomarkers of interest in both in vitro and in vivo settings. Detection is achieved by trapping the biomarker at the electrode tip. The trapping event alters the capacitive junction between a platinum wire capacitive immunoprobe and the surrounding conductive fluid environment, measured as a change in charge current in response to a potential shift in the probe. A unique electrical acquisition protocol was also presented that allows for "resetting" of the immunoprobe between detection cycles by electrostatically repelling the trapped biomarker from the probe tip. This biomarker clearance allows for subsequent cycles of measurement, thus providing a time-resolved measure¹⁰. In this implementation, the probe manufacture included electrodeposition of a polydopamine-reactive layer on the tip of a thin platinum wire electrode to which an antibody, raised against a biomarker of interest, is covalently bound. Dopamine and polydopamine differ in that polydopamine contains a quinone functional group. The quinones act as a target for nucleophilic attack by primary amines to form a covalent bond²¹. The most crucial step in this protocol is handling the physical wire with care, as any abrasion of the functionalized tip may render the CI probe unusable.

Additionally, ensuring CI probe stability prior to measurement is essential. It may be necessary to run several control standards in Tris-buffered saline prior to experimental testing; in doing so, the experimenter is looking for a stable transient curve in the resulting current (e.g., Figure 2D). Consistent fabrication practice can minimize this transient signal but not eliminate it.

The use of appropriate antibodies when investigating biomarkers of interest is another important factor when employing this technique. In this implementation, antibodies characterized as applicable in techniques detecting non-denatured proteins are best suited (e.g., IHC, ELISA, in situ hybridization) rather than those for denatured protein applications (western blot).

The use of immunochemical methods in the clinical diagnosis and assessment of heart disease progression, while well-established¹, relies primarily on samples derived from a tissue biopsy or sampling of the vascular compartment and, secondarily, on microdialysis-based interstitial fluid collection. Samples may require special preparation, handling, and technology to provide accurate results, which, at best, provide a measure of the biomarker of interest at minute-level temporal resolution^4,5. The capacitive immunoprobe methodology described provides several advantages over such approaches: 1) the probe manufacture and application described can be adapted to detect many unique biomarkers of significance; 2) the flexible platinum wire electrodes are deployable in the vasculature, in organs, or in other fluid compartments; 3) the signal derived provides time-resolved dynamic release profiles of biomarkers of interest in single subjects; 4) the CI probe maintains stability over time; 5) a stable recording condition can be maintained even in regions experiencing a high degree of mechanical stress or movement due to the flexibility of the sensor itself; and 6) the mobility of the system allows for dynamic readouts of biomarkers of interest at unprecedented resolution in vitro, in vivo, on the bench, and ultimately at the bedside.

The technique outlined above overcomes the spatiotemporal limitations inherent to traditional microdialysis-based biomarker detection and measurement. This methodology can assess dynamic release profiles on a near-instantaneous basis in single subjects without the need for pooled data or signal averaging over several experiments. The employment of this approach serves as a major technological advance in detecting and measuring relevant biomarkers for guiding clinical decision-making and therapeutic intervention.

Materials

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
Synthetic porcine NPY peptide	Bachem (Torrance, CA)	4011654