Antibody Conjugated Nanoparticle-based Quantification Using Dark Field Microscopy: A Procedure to Capture and Quantify Specific Exosomes from Body Fluids

Exosomes are small, membrane-enclosed structures that are secreted by the cells into the extracellular space. Exosomes are abundantly present in biofluids.

To identify and capture specific exosomes from body fluids, begin by taking a multi-well immunoassay slide. This slide is functionalized with proteins that act as antigens and bind to specific antibodies. 

Supplement the slide with the desired primary antibody solution and incubate. These antibodies bind to the pre-coated antigens on the slide surface.

Aspirate the remaining solution to remove any unbound antibodies. Now, add a blocking buffer that blocks the free antigens, thereby preventing the nonspecific binding of exosomes.

Then, remove the blocking buffer, load the exosomes containing serum suspension onto the slide, and incubate. This allows the exclusive binding of target-specific exosomes to the captured primary antibodies.

Next, load a suspension of gold nanoparticle conjugated secondary antibodies onto the slide surface. These antibodies attach to the pre-bound exosomes and form a detection complex.

Finally, using a pipette, discard any unbound secondary antibodies. Visualize the slides under a dark-field microscope. Gold nanoparticles present on the surface of exosomes scatter light and appear as bright spots against the dark background.  

To begin preparing the slide, dilute the desired exosome capture antibodies to 0.025 milligrams per milliliter in PBS. Pipette 1 microliter of the solution into each well of a protein A/G-treated slide backed with optical-grade glass. Then, transfer the slide to a humidifying box to ensure wells do not dry up during incubation.

Incubate the slide at 37 degrees Celsius for 1 hour to immobilize the capture antibodies. Then, aspirate the remaining solution to remove unbound antibodies. Wash the wells by adding and aspirating 1 microliter of PBS three times. Next, quickly load each well with 1 microliter of PBS-based blocking buffer, and incubate the slide at 37 degrees Celsius for 2 hours.

When running around 15 samples, we must use a single channel pipette to load working buffer onto 120 wells in less than 5 minutes to avoid the evaporation of blocking agent.

Start preparing a solution of antibody-conjugated gold nanorods during slide blocking. About 30 minutes before blocking finishes, rapidly thaw plasma or serum samples in a room temperature water bath. Vortex the thawed samples for 30 seconds to ensure that the suspensions are homogeneous. Then, centrifuge the samples at 500 x g for 15 minutes to precipitate protein aggregates and other debris.

Transfer 10 microliter aliquots of the supernatants to fresh tubes and make one-to-one dilutions with PBS. Mix the diluted samples by gentle vortexing or inversion as appropriate. When slide blocking has finished, aspirate the blocking buffer and wash the wells three times with 1 microliter portions of PBS. Immediately load the samples into the wells with 1 microliter per well and 8 replicates per sample.

Load exosome standards into the appropriate wells in the same way. Incubate the slide for 12 to 18 hours in a refrigerator at 4 degrees Celsius. Then, aspirate the wells and wash each well once with 1 microliter of PBS. Load 1 microliter of the antibody-conjugated gold nanorods suspension into each well and incubate the slide at 37 degrees Celsius for 2 hours.

Afterwards, aspirate the nanorod suspension and wash the slide in PBS supplemented with 0.01% Polysorbate-20 for 10 minutes using a mixer. Then, aspirate the wells and wash the slide in deionized water for 10 minutes on a rotating mixer. Remove the water and allow the slide to air-dry in a clean Petri dish before bringing the slide to the dark field microscope.