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

The overall goal of this procedure is to efficiently extract exosomes from biofluids, expel the exosome cargo, and rapidly analyze the cargo. This is accomplished by first extracting exosomes from biofluids using magnetic microparticles with exosome marker targeting antibodies, and then loading the extracted exosomes on an electrode surface. Next, the exosome is lyed using an electric field and the exosome cargo is hybridized to probes on the surface of the electrode.

A reporter molecule is also hybridized to the system. The final step is to add a substrate solution to the electrode surface and then obtain the readout by measuring the electric current. Ultimately, electrochemical measurement of current is used to show the amount of a nucleic acid or protein content in the exosome sample.

The main advantage of this technology over the existing method, such as the ultra centrification and the lysine buffer, is that it is a simple rapid method to extracting and testing exosome content. We are very excited about this technology because for the first time, this efim technology allow us to utilize the information that are shed by organs in disease or in normalcy that are shedded by the organ of origin through the vasculature and as they come into our oral cavity. This technology allow us to capture this information and release the molecular content that we can detect concurrently in a flexible fashion.

This disease discriminatory information, Generally, users new to this method may struggle because the usage of magnetic microparticles may be difficult if one is not careful. Furthermore, the usage of electrochemical methods in biological sample analysis may be a novel paradigm for some Visual demonstration of this method is critical as the magnetic micro particle and electro loading are difficult to learn. Special care must be taken when handling magnetic microparticle and load them onto electrolodes.

To begin resuspend, a stock solution containing 10 micrograms per milliliter of strept adin coated magnetic microparticles and add five microliters. The solution to a micro centrifuge tube containing 495 microliters of PBS to wash the beads. First, set the tube on a magnetic rack for one minute and carefully remove the buffer without disturbing the beads.

Then transfer the tube to a non-magnetic rack. Add 500 microliters of PBS and by PET to wash the beads. Do this a total of three times after the final wash reus.

Suspend the beads in 490 microliters of PBS and then add five microliters of Biotinylated mouse anti-human CD 63 antibody from a one milligram per milliliter stock solution. Mix the solution by pipetting program a sample rotator for reciprocal rotation at 90 degrees for five seconds and vibration at five degrees for one second. Place the tubes in the rotator and run the program for 30 minutes at room temperature.

Next, transfer the tubes to the magnetic rack for five minutes. Wash the beads three times with 500 microliters of PBS and then resuspend them in 490 microliters of PBS containing Caine. Label a tube with the sample identification and then add 10 microliters of the sample pipette several times to mix the sample with the antibody conjugated beads.

Next, transfer the tubes to the sample rotator and run the same program for two hours at room temperature. Then wash the bead three times with 500 microliters of a one molar tris hydrochloride buffer. Using the magnetized rack, the exosomes are now bound the magnetized beads to preco the electrode.

First dilute stock reagents of the probe, potassium chloride, and the ole monomer into distilled water. The five prime biotinylated DNA probe is specific for GA DH mRNA. Mix the monomer probe solution by vortexing.

Use a 16 sensor electrode array in which each unit electrode consists of working counter and reference electrodes composed of bare gold. Place a plastic well on the electrode array to prevent cross-contamination of the electrodes. Transfer 60 microliters of the monomer probe solution to the surface of a gold electrode so that the working counter and reference electrodes are covered by the liquid.

Apply a cyclic square wave electric field profile known as A-C-S-W-E field to electro polymerized a monomer probe. This creates a conducting polymer layer on the electrode surface. Rinse the sensor three times with the distilled water and then dry it with nitrogen gas to remove liquid from the surface of the electrode.

After preco the electrode with the capture probe, add five microliters of a one micromolar solution of the detector probe to 495 microliters of the bead exosome complex solution, and then mix by pipetting.Here. The probe is a three prime fluorescein conjugated DNA primer that is specific for GA dh. Placed a pre-coded electrode array over a magnet array consisting of 16 magnets aligned to the working electrodes of the sensor.

Then transfer 60 microliters of the bead exosome solution to the electrode surface. Apply A-C-S-W-E field for 20 cycles. The released exo somal cargo will hybridize to the probe on the electrode surface.

Rinse the electrode surface three times with distilled water to remove any unbound analytes, and then draw the electrode with nitrogen gas. Next, make a one to 1000 dilution of anti fluorescein HRP conjugated antibody in PBS containing Caine. Then add 60 microliters of the antibody dilution to the electrode surface.

Apply A-C-S-W-E field to the electrode surface to conjugate the anti fluorescein HRP antibody to the detector probe. Rinse the sensor surface three times with distilled water to remove excess antibody, and then draw the electrode with nitrogen gas. Next, load 60 microliters of TMB substrate onto each sensor surface with a multi-channel pipette using an electrochemical potentials stat measure the electrode current at negative 200 millivolts for 60 seconds.

To obtain the tric readout transmission electron microscopy was used to determine whether exosomes were bound to the magnetic beads. The 70 to 100 nanometer size granules attached to the beads are consistent with the known size of exosomes upon lysis by the electric field or detergent. No exosomes are apparent near the beads.

GA DH mRNA in exosomes was detected by atric readout. The amount of GA DH present over time decreases as expected upon exosome lysis by either the electric field or detergent, indicating exposure to the extra vesicular environment of the saliva. Efim was used to measure the exo somal content of the serum and the saliva of mice that were injected with human lung cancer cells producing a human CD 63 GFP Exo Somal marker.

After 20 days, higher levels of CD 63 GFP were found in both the serum and the sali of the mice injected with the cancer cells, but not to the saline injected mice. When attempting this procedure, it is important to remember, avoid touching the beads to extract adequate amount of liquid when washing the beads to wash the electrode surface uniformly, and to load the regions on the electrode properly. This technology, this efim technology, provided that critical unmet capability of how a systemic disease, a distal organ, can shed their information and calculate it in this entity called exosomes.

How they come into saliva, into our oral cavity and be able to capture the exosomes, release these molecular target, and allow it to be detected concurrently of like threatening diseases, diabetes, pancreatic cancer, or other life-threatening disorders. After watching this video, you should have a good understanding of how to rapidly extract exosomes from a biofluid, how to unload the cargo of the exosomes, and how to rapidly analyze the content of the exosomes using an integrated electrode system.