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, and then use a pipette tip to carefully remove the supernatant buffer without disturbing the beads.</li>
		<li>Place the tubes on a regular rack without magnets at the side. Add 500 &#181;l of PBS into the tubes, and use the pipette to mix the solution and beads together. Then put the tubes back on the magnetic rack to again separate the beads from the solution.</li>
		<li>Perform this removal of buffer via magnetization and resuspension in PBS a total of three times. This performs an initial wash of the magnetic particles.</li>
	</ol>
	</li>
	<li>Resuspend the beads into 490 &#181;l of PBS buffer, with the tube placed on the non-magnetized portion of the magnetic rack. Pipet 5 &#181;l of biotinylated mouse anti-human CD63 antibody at 1.0 mg/ml stock concentration into the mixture of beads. Use the pipette to mix the beads and antibody in solution.</li>
	<li>Place the microcentrifuge tubes with bead and biotinylated antibody mixture on a sample rotator. Set the rotator parameters for the sample rotator for reciprocal rotation at 90&#176; tilting for 5 sec and vibrating at 5&#176; for 1 sec. Rotate the sample-bead mixture tubes at these parameters for 30 min at RT.</li>
	<li>Remove unbound antibody after conjugation.
	<ol>
		<li>Following 30 min of rotation at RT, place the tubes back in the magnetic rack for 5 min.</li>
		<li>Perform three washes of beads by removing the liquid phase using a micropipette and wash with 500 &#181;l of PBS. After the triple wash, resuspend the beads in 490 &#181;l of casein-PBS and place on the unmagnetized portion of the rack.</li>
	</ol>
	</li>
	<li>Exosome extraction using antibody-coated beads.
	<ol>
		<li>Label each tube with targeted sample ID. Pipette a 10 &#181;l sample of serum or saliva into the microcentrifuge tube. Use the pipette to mix the sample and magnetic beads by pipetting several times.</li>
		<li>Place the tubes with sample and anti-human CD63 antibody beads on rotator and rotate for 2 hr at RT. Use the same rotator parameters as described in step 1.2.</li>
		<li>Following 2 hr of sample rotating, perform a triple wash by magnetizing to separate beads from solution, removing liquid phase with micropipette, and resuspending beads in 500 &#181;l of Tris-HCl buffer. The resultant beads are now bound to the exosomes and are ready for the electrical field release and measurement.</li>
	</ol>
	</li>
</ol>
2. Electric Field Induced Released and Measurement of Exosomal Content
<ol>
	<li>Initial Precoating of Electrode with GADPH Primer
	<ol>
		<li>Apply a plastic well to an electrode array to prevent cross-contamination of the individual electrodes. For this experiment, use a 16-sensor electrode array with each unit electrode in the array consisting of a working, counter, and reference electrode made of bare gold.</li>
		<li>Prepare a stock mixture of 100 nM DNA probe, 0.3 M KCl, and 10 mM pyrrole by pipetting stock reagents into a tube with ultrapure distilled water. Mix thoroughly by vortexing. 
		NOTE: For this study, the DNA probe selected corresponds to the GAPDH reference gene, which is known to exist within exosomes. The probe sequence used is: 5&#39;-Biotin-AGGTCCACCACTGACACGTTG-3&#39;. Use this mixture on all the electrodes.</li>
		<li>Pipet 60 &#181;l of the monomer-DNA probe mixture onto the surface of each gold electrode. Examine the electrodes to ensure that there is adequate coverage of the working, counter, and reference electrodes by the liquid mixture.</li>
		<li>Electropolymerize monomer-probe mixture to create a conducting polymer layer on the electrode surface by applying a cyclic square wave (CSW) electric field profile to the electrode surface. This electric field consists of applying +350 mV for 9 sec and immediately switching to +950 mV for 1 sec. Apply this cyclic square wave profile to the electrode for 10 cycles, for a total of 100 sec of applied electric field.</li>
		<li>Rinse sensor surface 3 times with distilled water and dry with nitrogen gas to remove liquid from the surface of the electrode. Ensure that liquid is properly removed from the electrode.</li>
	</ol>
	</li>
	<li>Exosome Cargo Unloading
	<ol>
		<li>Load 5 &#181;l of 1 &#181;M of a detector probe into 495 &#181;l of the bead-exosome complex mixture and use a pipette to mix. 
		NOTE: The detector probe is a DNA primer sequence conjugated to a fluorescein molecule at the 3&#39; end. The detector probe sequence used for this study corresponds to the GAPDH mRNA found within exosomes. The sequence of the fluorescein conjugated detector probe is: 5&#39;-GCAGTGGGGACACGGAAGGCC-Fluorescein-3&#39;.</li>
		<li>Pipette 60 &#181;l of the probe and bead-exosome complex mixture onto a gold electrode surface with a magnet array underneath. This magnet array consists of sixteen 2.54 mm diameter neodymium magnets aligned to correspond to the working electrodes of the sensor. Figure 2A&#160;illustrates the placement of the magnets and bead-exosome solution.</li>
		<li>Once sample is loaded on the electrode surface, apply 20 cycles of the CSW electric field with 9 sec at -300 mV and 1 sec at +200 mV (200 sec total). The exosomal cargo that is released will hybridize to the primers on the surface of the electrode. If surface markers of the exosome are the subject of investigation, skip this portion of the experiment. Figure 2B&#160;illustrates this process.</li>
		<li>Wash-off the unbound analytes on the electrode surface by triple rinsing the electrode surface with distilled water. Dry the electrode with nitrogen gas.</li>
	</ol>
	</li>
	<li>Reporter Antibody and Readout
	<ol>
		<li>Add 60 &#181;l of 150 unit/ml anti-fluorescein antibody conjugated to horseradish peroxidase (HRP in 1:1,000 dilution) diluted in Casein/PBS.</li>
		<li>Use an electric-field driven conjugation to complex anti-fluorescein HRP to the probe sandwich. Apply -200 mV for 1 sec and +500 mV for 1 sec for 5 cycles to the electrode surface. Figure 2A&#160;shows the capture and detector probe complexes for both a protein and nucleic acid system.</li>
		<li>Triple wash sensor surface using distilled water and dry with nitrogen gas.</li>
		<li>Following the wash-off of unbound excess anti-fluorescein antibody, add 60 &#181;l of 3,3&#39;,5,5&#39;-Tetramethylbenzidine (TMB) substrate. Load this substrate on to each sensor surface using a multichannel pipet.</li>
		<li>Perform amperometric readout of the current by measuring the electrode current at -200 mV for 60 sec using an electrochemical potentiostat capable of simultaneous measurement of 16 channels. Figure 2C&#160;is an example of current profile during readout.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/52439/52439fig2highres.jpg" />
Figure 2. Components of EFIRM Method. (A) Method of extracting exosomes from biofluid using anti-human CD63 coated magnetic microparticles and then unloading exosome cargo using cyclic square waves applied to the particle-exosome complex. (B) Scheme of electrode biosensor used for detecting RNA/DNA/protein targets from the released exosome. (C) Representative example of amperometric readout from the EFIRM methodology, where larger current magnitude corresponds to higher levels of a biomolecule. This figure is from Wei et al.14 <a href="https://www.jove.com/files/ftp_upload/52439/52439fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol>
	<li>Rabinowits, G., Ger&#231;el-Taylor, C., Day, J. M., Taylor, D. D., Kloecker, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomal+MicroRNA:+A+Diagnostic+Marker+for+Lung+Cancer.">Exosomal MicroRNA: A Diagnostic Marker for Lung Cancer.</a> Clinical Lung Cancer. 10 (1), 42-46 (2009).</li><li>Thakur, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-stranded+DNA+in+exosomes:+a+novel+biomarker+in+cancer+detection.">Double-stranded DNA in exosomes: a novel biomarker in cancer detection.</a> Cell Research. 24 (6), 766-769 (2014).</li><li>Lau, C. S., Wong, D. T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+Cancer+Exosome-like+Microvesicles+and+Salivary+Gland+Cells+Interplay+Alters+Salivary+Gland+Cell-Derived+Exosome-like+Microvesicles+In.">Breast Cancer Exosome-like Microvesicles and Salivary Gland Cells Interplay Alters Salivary Gland Cell-Derived Exosome-like Microvesicles In.</a> Vitro. PLoS ONE. 7 (3), e33037 (2012).</li><li>Bala, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs+in+exosomes+indicate+hepatocyte+injury+and+inflammation+in+alcoholic,+drug-induced,+and+inflammatory+liver+diseases.">Circulating microRNAs in exosomes indicate hepatocyte injury and inflammation in alcoholic, drug-induced, and inflammatory liver diseases.</a> Hepatolog. 56 (5), 1946-1957 (2012).</li><li>Dear, J. W., Street, J. M., Bailey, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+exosomes:+A+reservoir+for+biomarker+discovery+and+potential+mediators+of+intrarenal+signalling.">Urinary exosomes: A reservoir for biomarker discovery and potential mediators of intrarenal signalling.</a> Proteomics. 13 (10-11), 1572-1580 (2013).</li><li>L&#228;sser, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+saliva,+plasma+and+breast+milk+exosomes+contain+RNA:+uptake+by+macrophages.">Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages.</a> Journal of Translational Medicine. 9 (1), 9 (2011).</li><li>Palanisamy, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructural+and+Transcriptomic+Analyses+of+Human+Saliva+Derived+Exosomes.">Nanostructural and Transcriptomic Analyses of Human Saliva Derived Exosomes.</a> PLoS ONE. 5 (1), e8577 (2010).</li><li>Camussi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes/microvesicles+as+a+mechanism+of+cell-to-cell+communication.">Exosomes/microvesicles as a mechanism of cell-to-cell communication.</a> Kidney International. 78 (9), 838-848 (2010).</li><li>Lau, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+pancreatic+cancer-derived+exosomes+in+salivary+biomarker+development.">Role of pancreatic cancer-derived exosomes in salivary biomarker development.</a> The Journal of Biological Chemistry. 288 (37), 26888-26897 (2013).</li><li>Th&#233;ry, C., Amigorena, S., Raposo, G., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Exosomes+from+Cell+Culture+Supernatants+and+Biological+Fluids.">Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids.</a> Current Protocols in Cell Biology. 3 (22), (2001).</li><li>Park, N. J., Li, Y., Yu, T., Brinkman, B. M. N., Wong, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+RNA+in+Saliva.">Characterization of RNA in Saliva.</a> Clinical Chemistry. 52 (6), 988-994 (2006).</li><li>Wei, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio/Abiotic+Interface+Constructed+from+Nanoscale+DNA+Dendrimer+and+Conducting+Polymer+for+Ultrasensitive+Biomolecular+Diagnosis.">Bio/Abiotic Interface Constructed from Nanoscale DNA Dendrimer and Conducting Polymer for Ultrasensitive Biomolecular Diagnosis.</a> Small. 5 (15), 1784-1790 (2009).</li><li>Wei, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+Sensor+for+Multiplex+Biomarkers+Detection.">Electrochemical Sensor for Multiplex Biomarkers Detection.</a> Clinical Cancer Research. 15 (13), 4446-4452 (2009).</li><li>Wei, F., Yang, J., Wong, D. T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+exosomal+biomarker+by+electric+field-induced+release+and+measurement+(EFIRM).">Detection of exosomal biomarker by electric field-induced release and measurement (EFIRM).</a> Biosensors and Bioelectronics. 44, 115-121 (2013).</li></ol>

David Wong is co-founder of RNAmeTRIX Inc., a molecular diagnostic company. PeriRx LLC sublicensed intellectual properties pertaining to molecular diagnostics from RNAmeTRIX. David Wong is a consultant to PeriRx.

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.

<table border="0" cellpadding="0" cellspacing="0" width="802">
	<tbody>
		<tr height="21">
			<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 ...

detection-exosomal-biomarker-electric-field-induced-release

Research

JoVE Journal

Bioengineering

18.3K Views.  University of California, Los Angeles. 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. 

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

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is subject to chemical and mechanical signals, and the mechanisms by which it senses and responds to mechanical signals are poorly understood. Elucidating the role of forces in cell maturation will enable the design of scaffolds that can promote cell adhesion and cytoskeletal coupling to the substrate, and therefore improve the capacity of different neuronal types to regenerate after injury.
Here, we describe a method to apply simultaneous force spectroscopy measurements during laser induced cell lesion. We measure tension release in the partially lesioned axon by simultaneous interferometric tracking of an optically trapped probe adhered to the membrane of the axon. Our experimental protocol detects the tension release with piconewton sensitivity, and the dynamic of the tension release at millisecond time resolution. Therefore, it offers a high-resolution method to study how the mechanical coupling between cells and substrates can be modulated by pharmacological treatment and/or by distinct mechanical properties of the substrate.

We measured the tension release in an axon that was partially lesioned with a laser dissector by simultaneous force spectroscopy measurement performed on an optically-trapped probe adhered to the membrane of the axon. The developed experimental protocol evaluates the axon adhesion to the culture substrate.

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Fabrication of a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water samples. A brief overview of the evolution of chip-based SPE techniques is provided. This is followed by an introduction to specific polymeric materials and their role in SPE. To develop an innovative dipole-assisted SPE technique, a chlorine (Cl)-containing SPE functionality was implanted into a poly(methyl methacrylate) (PMMA) microchip. Herein, diverse analytical techniques including contact angle analysis, Raman spectroscopic analysis, and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis were employed to validate the utility of the implantation protocol of the C-Cl moieties on the PMMA. The analytical results of the X-ray absorption near-edge structure (XANES) analysis also demonstrated the feasibility of the Cl-containing PMMA used as an extraction medium by virtue of the dipole-ion interactions between the highly electronegative C-Cl moieties and the positively charged metal ions.

The fabrication protocol of a dipole-assisted solid phase extraction microchip for the trace metal analysis is presented.

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...