Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

Summary

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

Abstract

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of exosomes to determine if they carry disease discriminatory biomarkers. For performing exosomal analysis, it is necessary to develop a method for extracting and analyzing exosomes from target biofluids without damaging the internal content.

Electric field-induced release and measurement (EFIRM) is a method for specifically extracting exosomes from biofluids, unloading their cargo, and testing their internal RNA/protein content. Using an anti-human CD63 specific antibody magnetic microparticle, exosomes are first precipitated from biofluids. Following extraction, low-voltage electric cyclic square waves (CSW) are applied to disrupt the vesicular membrane and cause cargo unloading. The content of the exosome is hybridized to DNA primers or antibodies immobilized on an electrode surface for quantification of molecular content.

The EFIRM method is advantageous for extraction of exosomes and unloading cargo for analysis without lysis buffer. This method is capable of performing specific detection of both RNA and protein biomarker targets in the exosome. EFIRM extracts exosomes specifically based on their surface markers as opposed to size-based techniques.

Transmission electron microscopy (TEM) and assay demonstrate the functionality of the method for exosome capture and analysis. The EFIRM method was applied to exosomal analysis of 9 mice injected with human lung cancer H640 cells (a cell line transfected to express the exosome marker human CD63-GFP) in order to test their exosome profile against 11 mice receiving saline controls. Elevated levels of exosomal biomarkers (reference gene GAPDH and protein surface marker human CD63-GFP) were found for the H640 injected mice in both serum and saliva samples. Furthermore, saliva and serum samples were demonstrated to have linearity (R = 0.79). These results are suggestive for the viability of salivary exosome biomarkers for detection of distal diseases.

Introduction

Exosome research is an emerging field of investigation that examines lipid microvesicles that carry RNA¹, DNA², and protein³ cargo. Previous investigations of exosome biology have led to identification of exosomes in biofluids such as blood⁴, urine⁵, breast milk⁶, and saliva⁷. Studies have demonstrated that exosomes play a role in different cellular pathways, remotely meditating communication between different systems of the body⁸. Because of the role exosomes play in intercellular communication, it is hypothesized that they may package biomolecule targets (protein, RNA, and DNA) c....

Protocol

1. Magnetic Bead-based Exosome Extraction

1. Pipette a well-mixed solution of 5 µl of Streptavidin-Coated magnetic microparticles into 495 µl of phosphate buffered saline (PBS) buffer in a microcentrifuge tube to resuspend the beads. Wash and resuspend the beads with 500 µl of PBS three times using a magnetic rack. The rack is an array of magnets on the side of a housing unit that can hold the sample microcentrifuge tubes.
   1. For each wash, first let the tubes sit on the rack for 1 min, and then use a pipette tip to carefully remove the supernatant buffer without disturbing the beads.
   2. Place the tubes on a regular rack without magnets at th....

Results

Validation of Exosome Capture of Beads Using TEM

Isolation of exosomes from saliva using anti-human CD63 magnetic beads was validated following extraction protocol by using transmission electron microscopy (TEM) images. TEM shows magnetic beads with 70-100 nm granules immediately adjacent (see Figure 3A, and 3B), consistent with the known profile of exosomes. No 70-100 nm granules were observed for the magnetic beads in saliva that did not hav.......

Discussion

As the results indicate, anti-human CD63 coated magnetic nanoparticles are able to specifically capture small particles that have a size ranging from 70-100 nm. This captured particle is consistent with the previously observed profile of exosomes. Furthermore, the usage of the low-voltage CSW following the capture of the particles is shown to remove them from the bead surface and cause DNA degradation profiles similar to that of a traditional lysis buffer based method for cargo release. This data indicates that the workf.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Helios 16-Channel Reader System with Chip Interface	Genefluidics, USA	RS-1000-16
16x Sensor Chip, Bare Gold, pack of 5 chips	Genefluidics, USA	SC1000-16X-B
Biotinylated anti-human CD63 Antibody	Ancell, USA	215-030
Dynabeads MyOne Streptavidin T1	Invitrogen, USA	65601
Neodynium Magnetics (1/10" dia. x 1/32" thick)	K&J Magnetics, USA	DH101
Ultrapure Distilled Water	Life Technologies, USA	10977-023
Mettler Toldeo 3 M KCl Solution	Fisher Scientific, USA	1911512
Pyrrole	Sigma-Aldrich, USA	W338605-100g
Anti-Fluorescein-POD, Fab fragments	Roche, Germany	11426346910
3,3′,5,5′-tetramethylbenzidine substrate (TMB/H2O2, low activity)	Neogen, Usa	330175
Phosphate Buffered Saline Solution	Life Technologies, USA	10010023
Casein/PBS	Fisher Scientific, USA	37532