We thank Dr. Olu Ajijola (UCLA Cardiac Arrhythmia Center) for expert support for the in vivo experiments. This work was supported by NIH U01 EB025138 (JLA, CS).

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
<ol>
	<li>Cut a 25 cm length of perfluor...

<ol>
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H., von R&#252;den, L., Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-Channel Recording, Second Edition. , 245-275 (1995).</li><li>Ren, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile,+high+efficiency+immobilization+of+lipase+enzyme+on+magnetic+iron+oxide+nanoparticles+via+a+biomimetic+coating.">Facile, high efficiency immobilization of lipase enzyme on magnetic iron oxide nanoparticles via a biomimetic coating.</a> BMC Biotechnology. 11, 63 (2011).</li></ol>

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 e...

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 compartment1,2,3 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 system6,7,8,9. 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 tip11,12. 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 probe10,13.
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 &#34;reset&#34; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AgCl disc electrode</td>
			<td>Warner Instruments (Holliston, MA)</td>
			<td>64-1307</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-NPY monoclonal antibody</td>
			<td>Abcam, (Cambridge, MA)</td>
			<td>ab112473</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom multichannel amplifier/ 1 M&#937; feedback resistor multichannel headstage</td>
			<td>NPI Electronic, (Tamm, Germany)</td>
			<td>NA</td>
			<td>Based on NPI VA-10M multichannel amplifier</td>
		</tr>
		<tr>
			<td>Dopamine HCl</td>
			<td>Sigma Aldrich (St. Louis, MO)</td>
			<td>H8502-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold-plated male connector pin</td>
			<td>AMP-TE Connectivity (Amplimite)</td>
			<td>6-66506-1</td>
			<td></td>
		</tr>
		<tr>
			<td>HEKA LIH 8+8 analog-to-digital/digital-to-analog device</td>
			<td>HEKA Elektronik, (Holliston, MA)</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Igor Pro data acquisition software, v. 7.08</td>
			<td>WaveMetrics, (Lake Oswego, OR)</td>
			<td></td>
			<td>Software driving command potential and data acquisition was custom written</td>
		</tr>
		<tr>
			<td>Masterflex L/S Standard Digital peristaltic pump</td>
			<td>Cole Palmer, (Vernon Hills, IL)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PFA-coated platinum wire</td>
			<td>A-M Systems, (Sequim, WA)</td>
			<td>773000</td>
			<td>0.005&#8221; bare diameter, 0.008&#8221; coated diameter</td>
		</tr>
		<tr>
			<td>Silicone elastomer</td>
			<td>World Precision Instruments (Sarasota, FL)</td>
			<td>SYLG184</td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic porcine NPY peptide</td>
			<td>Bachem (Torrance, CA)</td>
			<td>4011654</td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic porcine NPY peptide</td>
			<td>Bachem (Torrance, CA)</td>
			<td>4011654</td>
			<td></td>
		</tr>
	</tbody>
</table>

time-resolved in vivo measurement of neuropeptide dynamics by capacitive immunoprobe in porcine heart

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.

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 c...

Watch this Scientific Journal Video about Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart at JoVE.com

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

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.

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

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical ...

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.

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1.9K visualizações.  Case Western Reserve University. Esta é a primeira vez que um método é desenvolvido para a medição de tempo resolvido dos níveis do transmissor de peptídeos in vivo. Podemos medir os níveis do transmissor de peptídeos em indivíduos únicos e locais discretos com análises quase em tempo real. Primeiro, corte um comprimento de 25 centímetros de fio de platina revestido de perfluoroalkoxy.Em seguida, usando um bisturi, retire aproximadamente cinco milímetros de revestimento perfluoroalkoxy de uma extremidade...