The overall goal of this procedure is to isolate extracellular vesicles using a paper-based platform. This is accomplished by first laminating with two polystyrene sheets with circular cellulose, paper cutouts aligned on an array of registered through holes. The second step is to chemically conjugate molecules that would bind extracellular vesicles to the paper test zone.
Next, the biological sample can be pipetted directly onto the paper test zone, and extracellular vesicles are isolated after the rinse step. The final step is to characterize, captured extracellular vesicles using electron microscopy, Eliza or transcriptomic analysis. Ultimately, subsets of extracellular vesicles from as small as 10 microliter serum samples can be purified and concentrated.
So the main advantage of this technique over existing methods like ultra education is that it's much gentle assemble and it's simple to perform. Demonstrating the procedure will be a grad student shall you. Starting with two sheets of polystyrene, create an array of circular cutouts.
Each exhibiting a diameter of less than five millimeters with either a manual hole punch or cad, automated cutter plotter. In addition, cut out a series of five millimeter diameter chromatography, paper discs with a hole punch. Place one polystyrene sheet on the bench and overlay a single paper disc on top of each corresponding through hole.
Sandwich the paper discs by registering and overlaying the second sheet of polystyrene. Check the overall alignment before bonding the sandwich in a thermal laminator. Next, begin chemical surface modifications on the paper discs by inserting the laminated device into a plasma chamber.
Briefly activate the paper surface with oxygen plasma upon release of chamber vacuum immediately transfer the paper device into the cylin solution and incubate for 30 minutes. Set room temperature. Remove excess cy lanes by rinsing the device with five milliliters of 200 proof ethanol.
Then incubate the device with A-G-M-B-S linker solution for 15 minutes. Set room temperature. Remove excess linker molecule with copious amounts of ethanol rinses.
Next, incubate the device with avadon for one hour at four degrees Celsius and remove unbound avadon with a five milliliter PBS wash. The device can then proceed to either the antibody binding step or be stored at four degrees Celsius for four weeks if needed. Upon Aden attachment pipette 10 microliters of blocking solution onto the exposed paper disc regions and incubate the paper for 10 minutes.
At room temperature, repeat the addition of 10 microliters of blocking solution and the 10 minute room temperature incubation. Two more times to complete the bio functionalization of the exposed paper substrate pipette 10 microliters of the biotinylated capturing molecules onto the discs and incubate for 10 minutes At room temperature for extracellular vesicles, the capturing molecules of choice can be either anti CD 63 antibodies or an xin five. Repeat the 10 microliter pipetting and the 10 minute incubation processes.
Two more times After capture molecule attachment. Wash off excess moieties by pipetting. 10 microliters of the appropriate wash buffer onto each paper disc.
Incubate the wash for one minute at room temperature and repeat the 10 microliter wash and one minute incubation steps two more times. The fully functionalized paper device is now ready for isolating extracellular vesicles from serum or aqueous humor For serum processing. Collect 10 milliliters of peripheral blood in a vacutainer tube and gently invert the tube five times.
Place the tube in a vertical position and wait for 30 minutes. Set room temperature, centrifuge the blood sample for 15 minutes. Set room temperature.
Transfer the top clear serum layer to a clean plastic tube and discard the lower semi-solid phase. Clarify the serum at a higher centrifugation speed for 30 minutes. Set room temperature.
Transfer the supernatant into a fresh tube and remove fine particulates by passing the supernatant through a 0.8 micrometer filter. The resulting filtered serum can be used immediately or be stored at negative 80 degrees Celsius. If the extracellular vesicles are to be extracted from humor, collect aqueous humor from patients directly through invasive procedures and store the samples at negative 80 degrees Celsius.
Until use, no filtration or clarification steps are needed for vitreous humor samples. To isolate extracellular vesicles from either serum or aqueous humor, spot pipette five microliters of the biological sample onto each bio functionalized paper substrate disc. Incubate the spot for one minute at room temperature and repeat the five microliter pipetting and the one minute incubation processes.
Two more times After affinity binding, rinse off unbound vesicles by first pipetting 10 microliters of the appropriate wash buffer. Incubate the buffer for one minute at room temperature and repeat the 10 microliter wash and the subsequent incubation. Step two more times.
The captured extracellular vesicles are now ready for the following downstream assays to fix the captured vesicles onto the functionalized paper support pipette 10 microliters of 0.5 x karnofsky fixative onto each paper disc. Let the cross-linking reaction proceed at room temperature for 10 minutes and rinse all discs with copious amounts of PBS for five minutes. Next, dehydrate the paper substrates by applying the following washes in order.
One 35%ethanol incubation, two 50%ethanol incubations, two 70%ethanol incubations, two 95%ethanol incubations, and four 100%ethanol incubations. For each individual wash, let the ethanol solution incubate on top of the paper discs at room temperature for 10 minutes. When the paper device is bone dry sputter coat the samples with an 18 nanometer layer of palladium gold in a metal sputter.
Insert the sample into a scanning electron microscope and perform microscopy at five kilovolts to prevent excess charging of the biological sample to detect captured vesicles via immunoassays. Start by adding five microliters of anti CD nine primary antibody onto each paper disc and incubate at room temperature for one minute. Remove all unbound antibodies by washing the paper device with 30 milliliters of room temperature PBS via mechanical agitation.
Next pipette five microliters of horse radish peroxidase conjugated secondary antibody onto each paper. Disc and incubate at room temperature for one minute. Wash the device with 30 milliliters of PBS via mechanical agitation to remove unbound secondary antibodies.
Finally, add five microliters of color and metric substrate mixture onto each paper.Disc. Incubate the reaction at room temperature for 20 to 30 minutes and scan the resulting change in spot color intensity using a desktop scanner. To begin, isolate individual paper discs from the bulk device by cutting away regions of excess polystyrene laminate using scissors or a razor blade.
Place each paper disc into a clean 1.5 milliliter micro centrifuge. Two lys the captured vesicles by adding 265 microliters of lysis buffer. And 35 microliters of PVP based polysaccharide removal carrier to each tube vortex vigorously.
To ensure complete vesicle, lysis and dislodgement of liberated RNA molecules, add 30 microliters of homogenate solution to the lysate vortex to mix and incubate the tube on ice for 10 minutes. Next, add 330 microliters of acid phenol chloroform mixture to the lysate to ensure proper phase specific separation of RNA from both proteins and DNA vortex vigorously for at least 15 seconds per tube. Centrifuge the tube at full speed for five minutes.
At room temperature, transfer the RNA containing top aqueous phase into a fresh 1.5 milliliter tube and discard the lower organic phase. Add 1.25 aqueous volumes worth of 100%ethanol to the RNA and vortex briefly to mix. Then apply the mixture onto a commercial RNA solid extraction column.
Centrifuge the tube for 15 seconds at room temperature and discard the flow through fraction. Wash the column by adding 700 microliters of working solution. Number one, centrifuge the tube for five to 10 seconds and discard the flow through.
Next, wash the column with 500 microliters of working solution. Number two, centrifuge the tube for five to 10 seconds. Discard the flow through and repeat the wash one more time.
Remove any residual ethanol with a final one minute spin down at room temperature and transfer the dried extraction column into a fresh 1.5 milliliter tube. Add 100 microliters of preheated R RNA free water. Centrifuge the tube at full speed for five to 10 seconds and discard the top extraction column if deemed necessary.
Concentrate and further purify the RNA EIT using a commercial RNA cleanup kit using fluorescently labeled biotin. As a reporter for surface Im mobilization. The efficacy and specificity of the paper functionalization protocol can be seen by comparing the high fluorescence incurred on the crosslinker modified surface over non functionalized paper surfaces.
Moreover, when the reporter molecule is replaced by a biotinylated anti CD 63 antibody, the properly functionalized paper substrate will facilitate a higher extracellular vesicle capture rate over a negative control paper substrate. When different capturing molecules are immobilized onto paper substrates, different subgroups of extracellular vesicles appear to exhibit different size distributions using the paper device. One can also assay for both the intravesical RNA content and the abundance of vesicle specific surface proteins.
After watching this video, you should have a good understanding of how to isolate and cater rise at extracellular les using a paper-based device.
