Antibody Conjugated Nanoparticle-based Quantification Using Dark Field Microscopy: A Procedure to Capture and Quantify Specific Exosomes from Body Fluids
Published: April 30, 2023
Abstract
Source: Wan, M. et al. Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes. J. Vis. Exp. (2019)
This video describes the use of low-magnification, dark-field microscopy to quantify specific exosomes in small volume biofluidic samples. The exosomes thus identified can serve as potential cancer biomarkers.
Protocol
1. Preparation of Nanoparticle Probes NOTE: This assay utilizes Functionalized Gold Nanorods (AuNRs; 25 nm diameter x 71 nm length) that are covalently conjugated with neutravidin polymers (AV) and have a surface plasmon resonance peak that produces a red (641 nm peak) scattering signal upon DFM illumination. Wash 40 µL of AuNR-AV (2.56 x 1011 particles) three times with 200 µL PBS (pH 7.0) by centrifugation and aspiration…
Divulgaciones
The authors have nothing to disclose.
Materials
| Eppendorf Repeater stream | Fisher Scientific | 05-401-040 | |
| Eppendorf Research plus | Eppendorf | 3120000011 | 0.1 – 2.5 µL, dark gray |
| Functionalized Gold Nanorods | Nanopartz | C12-25-650-TN-DIH-50-1 | In vitro neutravidin polymer functionalization |
| HulaMixer Sample Mixer | Thermo Fisher Scientific | 15920D | |
| Incu-shaker 10L | Benchmark Scientific | H1010 | |
| Inverted Research Microscope | Nikon | Ti-DH | With Dark field condenser, DS-Ri2 camera, and Ti-SH-U universal holder, and motorized stage |
| NIS-Elements | Nikon | Microscope imaging software | |
| Phosphate Buffered Saline (1X) | GE Healthcare Life Sciences | SH30256.02 | HyClone |
| Protein A/G Treated Glass Substrate Slides |
Arrayit Corp. | AGMSM192BC | Premium microarray substrate |
| Q500 Sonicator | Qsonica, LLC | Q500-110 | With standard probe (#4220) |
| Superblock blocking buffer | Thermo Scientific | ||
| TWEEN 20 | Sigma Life Sciences | 9005-64-5 |
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Antibody Conjugated Nanoparticle-based Quantification Using Dark Field Microscopy: A Procedure to Capture and Quantify Specific Exosomes from Body Fluids. J. Vis. Exp. (Pending Publication), e20498, doi: (2023).