The complex nature of EVs makes it difficult to recognize the subpopulation of interest, and this protocol can detect them with their phenotype, size profile, and nanomechanical properties. This combination of technique is a label-free method, which allows us to detect EVs real time from the condition media and blood plasma. This technique enables to characterize deeply subset of nanoparticles of interest, in order to be used as bioindicators of pathology, or also used as nanoparticle for drug delivery.
Since it is a very sensitive method, it is imperative to pay attention to the biochip preparation before injecting your sample of interest. After coating and functionalizing the chips, incubate the biochip in a mixture of 200-millimolar ethyl dimethylaminopropyl carbodiimide/N-hydroxysuccinimide and 50 millimolar per liter N-hydroxysuccinimide for at least 30 minutes in the dark at room temperature. Using the spotter, add 300 nanoliters of ligand solution and incubate the biochip under a sonic bath for 30 minutes.
Wash the biochip from the top with ultrapure water. Add a droplet of oil with the same refractive index as the prism to create a uniform thin layer between the biochip and the prism, and gently place it on a prism with the same refractive index as the biochip. For surface plasmon resonance imaging, mount the biochip on the SPRi system.
Navigate to the dropdown menu on the left side of the software, and click on the Working directory. Find the image where different spots are visible, and click to select this image. Then write the name of the ligand families, and click on the Finish species definition.
Drag the black line with the cursor to the optimum working angle. Click on Move mirror to working angle, and select a working angle. Now click on Kinetics.
As the software prompts the user to define the negative control, choose No negative control at this point. Inject rat serum albumin at 50 microliters per minute for four minutes. Inject ethanolamine at 20 microliters per minute for 10 minutes to deactivate the carboxylic groups still present and reactive on the surface.
Afterward, wash the biochip by injecting 40 millimolar OG at 50 microliters per minute for four minutes. Inject the extracellular vesicles at a specific concentration on the biofunctionalized chip while following the kinetics of the interaction of vesicles on the different spots, simultaneously. Also determine the value of reflectivity and the level of interaction.
Once stabilized, inject glutaraldehyde on the chip to fix extracellular vesicles at the place they are before doing AFM imaging. Align the biochip on the top of the mask on the glass slide. Use the CCD camera on top of the AFM to localize the cantilever on the correct spot that must be scanned.
Start the AFM acquisition in contact mode from three to five large to small areas. Next, treat the AFM images with JPK data processing software by first selecting the height channel. Choose a polynomial fit to be subtracted from each line to obtain straightened scan lines.
Select the height threshold on gold grains to eliminate the roughness on the surface. The grain extraction module marks the grains using a height threshold of 8.5 nanometers. Multiplexed biochips were analyzed after albumin passivation.
The chip with no default. The chip with some defects, due to fusion of spots, weak grafting, or bubbles or contaminants. And a naked gold chip without microarrays for examining the adsorption of extracellular vesicles on gold is shown.
The capture experiment on a multiplexed biochip showed good reflectivity signals for different ligands, and a good signal-to-noise ratio for the different ligands, since the response of the negative control was negligible. Extracellular vesicle adsorption after the injection of the extracellular vesicle sample displayed high reflectivity signal, suggesting those vesicles were able to adsorb and remain stable on the gold chip. After extracellular vesicle loading, the large-scale and small-scale AFM images of extracellular vesicles on biochips were generated.
A high-resolution closer view enables the metrology of extracellular vesicles. The effective diameter of the extracellular vesicles on a biochip ranged from 30 to 300 nanometers, with a large majority around 60 nanometers. The results obtained in air and liquid were comparable.
The Raman spectrum of extracellular vesicles adsorbed on a naked chip revealed a clear spectrum, with peaks corresponding mainly to methylene vibrations, which are associated with the lipids of extracellular vesicle membranes. It is important to prepare your biochip rigorously in order to be able to detect your EVs accurately and in a reproducible way. Conventional methods like Western blotting and nanoparticle tracking analysis can be engaged to correlate and confirm phenotypes and size patterns.
Moreover, multi-omic analyses are envisioned to even go deeper into EV molecular characterization and potential subset discrimination.
