Antibody Conjugated Nanoparticle-based Quantification Using Dark Field Microscopy: A Procedure to Capture and Quantify Specific Exosomes from Body Fluids

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.

1. Wash 40 µL of AuNR-AV (2.56 x 10¹¹ particles) three times with 200 µL PBS (pH 7.0) by centrifugation and aspiration (8,500 x g at 4 °C for 10 minutes), followed by a final centrifugation and aspiration step after which the AuNR-AV pellet is ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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