Why does nanoparticle-based light scattering matter for exosome quantification?

Gold nanoparticle-conjugated antibodies enhance light scattering from captured exosomes, enabling their visualization as bright spots under dark-field microscopy. This signal amplification allows specific exosome detection in complex biofluids without fluorescent labels. The method supports quantitative analysis by correlating spot intensity or count with exosome concentration.

How does antibody immobilization on immunoassay slides support target validation?

Capture antibodies are immobilized on protein A/G-treated slides to bind specific exosome surface markers, enabling selective isolation from serum. This step ensures that only target-specific exosomes are retained for downstream detection. The approach supports target validation by confirming the presence and abundance of disease-associated exosome populations.

What quantitative measurements enable exosome enumeration in this workflow?

Exosomes are quantified by counting bright spots generated from gold nanoparticle scattering under dark-field microscopy. Each spot corresponds to a single exosome bound to the slide via antibody-nanoparticle complexes. The method provides a direct, label-free readout for exosome concentration in microliter-scale biofluid samples.

Why are replication and washing steps critical for assay reliability?

Multiple washing steps remove unbound antibodies and reduce nonspecific binding, ensuring that only specifically captured exosomes contribute to the signal. Eight replicates per sample improve statistical confidence and account for well-to-well variability. These steps are essential for reproducible exosome quantification across experimental conditions.

What statistical analysis is required before implementing this assay in biomarker studies?

Implementation requires baseline signal normalization, replicate averaging, and threshold setting for positive exosome detection. Statistical comparison across conditions depends on spot count or intensity distributions from control and treatment groups. The method supports parametric or non-parametric tests based on replicate data quality and distribution assumptions.

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

JoVE Encyclopedia of Experiments

Cancer Research

0 views • 4:43 min • April 30th, 2023

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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.

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Last updated: 11 July 2026

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

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