Name: detection of exosomal biomarker by electric field-induced release and measurement (efirm)
Uploaded: 2015-01-23T15:00:00
Description: Watch this Scientific Journal Video about Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM at JoVE.com

This work was supported by the National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant UL1TR000124 (to FW); the Felix &#38; Mildred Yip Endowed Professorship and the Barnes Family Fund (to DTWW), the National Institute Of Dental &#38; Craniofacial Research of the National Institutes of Health under Award Number T90DE022734 (to MT). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Magnetic Bead-based Exosome Extraction
<ol>
	<li>Pipette a well-mixed solution of 5 &#181;l of Streptavidin-Coated magnetic microparticles into 495 &#181;l of phosphate buffered saline (PBS) buffer in a microcentrifuge tube to resuspend the beads. Wash and resuspend the beads with 500 &#181;l 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.
	<ol>
		<li>For each wash, first let the tubes sit on the rack for 1 min, a...

<ol>
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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...

An erratum was issued for <a href="http://www.jove.com/video/52439">Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)</a>. The disclosures were updated.

An erratum was issued for <a href="http://www.jove.com/video/52439">Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)</a>.

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

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) correlated with disease states. In vitro3 and animal model9 studies appear to corroborate this hypothesis. In investigating exosomal content for biomarker discovery, it is necessary to develop a methodology for selective exosome isolation from biofluids, induced expulsion of cargo from exosomes, and quantification of exosome biomolecules. In the extent of this work, exosomes will be defined as a structure having a diameter of approximately 70-100 nm and possessing surface marker CD63.
Researchers typically first purify exosomes by ultracentrifugation10 and then process exosomal content through the usage of lysis buffer kits. Usage of lysis buffer methods requires incubation times ranging from minutes to hours. This process may potentially harm exosome cargo and lead to sample degradation. For example, salivary exosome RNA released via lysis buffer into the surrounding extracellular environment possesses a half-life of under 1 min, making measurement of exosomal RNA post-lysis buffer a particularly difficult task without the addition of stabilization reagents11. The compounded effect of adding various reagents for lysis and stabilization may introduce agents that complicate and interfere with the analysis of exosomal content. An alternative approach may be helpful for rapidly unloading exosomal content and safely preserving the cargo for characterization.
In this work, we propose the usage of a non-uniform electric field for the release of exosomal content. Electric-fields have been known to carry the ability to polarize and disrupt the lipid bilayer that forms cell membranes. Our experimental work explores usage of non-uniform cyclic square waves (CSW) for disrupting the microvesicle structure of exosomes and releasing carried cargo. This method uses voltages in the several hundred millivolt range, meaning that most biomolecules will not be disrupted. We demonstrate that the usage of a cyclic-square wave is able to actuate release of salivary exosome mRNA content into the surrounding fluidic environment. This release of exosomal content is seamlessly integrated with an electrode system that can be used to quantify the biomarker expression levels12,13. This proposed method allows for rapid, sensitive, and lysis buffer free analysis of exosome content.
<img alt="Figure 1" src="/files/ftp_upload/52439/52439fig1highres.jpg" /> 
Figure 1. Overview of EFIRM Workflow. .The EFIRM method is broadly divided into the three major phases that are necessary for purifying and analyzing exosomes. 
This CSW based exosomal content release and analysis method is used in conjunction with CD63-specific magnetic microbeads for exosome isolation. These CD63-affinity beads allow for the selective isolation of exosomes from salivary samples (and other biofluids). Following incubation and extraction of exosomes using the magnetized beads, the beads are migrated to the electrochemical sensor system for the CSW based content release and analysis portion of the experiment. Figure 1 gives an overview of the workflow of the EFIRM method.

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			<td height="21" style="height:21px;width:347px;">Name of Material/ Equipment</td>
			<td style="width:157px;">Company</td>
			<td style="width:132px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Helios 16-Channel Reader System with Chip Interface&#160;</td>
			<td style="width:157px;">Genefluidics, USA</td>
			<td style="width:132px;">&#160;RS-1000-16</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">16X Sensor Chip, Bare Gold, pack of 5 chips</td>
			<td style="width:157px;">Genefluidics, USA</td>
			<td>SC1000-16X-B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Biotinylated anti-human CD63 Antibody</td>
			<td style="width:157px;">Ancell, USA</td>
			<td style="width:132px;">215-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Dynabeads MyOne Streptavidin T1</td>
			<td>Invitrogen, USA</td>
			<td>65601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Neodynium Magnetics ( 1/10&#34; dia. x 1/32&#34; thick)</td>
			<td style="width:157px;">K&#38;J Magnetics, USA</td>
			<td>DH101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">Ultrapure Distilled Water</td>
			<td style="width:157px;">Life Technologies, USA</td>
			<td>10977-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Mettler Toldeo 3M KcL Solution</td>
			<td style="width:157px;">Fisher Scientific, USA</td>
			<td>1911512</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Pyrrole</td>
			<td style="width:157px;">Sigma-Aldrich, USA</td>
			<td style="width:132px;">W338605-100g</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Anti-Fluorescein-POD, Fab fragments</td>
			<td style="width:157px;">Roche, Germany</td>
			<td>11426346910</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:347px;">3, 3&#8242;, 5, 5&#8242; tetramethylbenzidine substrate (TMB/H2O2, low activity)&#160;</td>
			<td style="width:157px;">Neogen, Usa</td>
			<td style="width:132px;">330175</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">Phosphate Buffered Saline Solution</td>
			<td style="width:157px;">Life Technologies, USA</td>
			<td align="right" style="width:132px;">10010023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Casein/PBS</td>
			<td style="width:157px;">Fisher Scientific, USA</td>
			<td>37532</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of exosomal biomarker by electric field-induced release and measurement (efirm)

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.

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...

Watch this Scientific Journal Video about Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM at JoVE.com

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

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.

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

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of ...

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