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Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart
In This Article
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 compartment1,2,3 by bulk fluid draw or the interstitial compartment by microdialysis. Microdialysis employs a semipermeable membrane tube of known length that is place....
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
- Cut a 25 cm length of perfluor.......
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 c.......
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 e.......
Acknowledgements
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).
....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 |
References
- Chow, S. L., et al. Role of Biomarkers for the prevention, assessment, and management of heart failure: A scientific statement from the American Heart Association. Circulation. 135 (22), 1054-1091 (2017).
- Goldstein, D. S.
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