Fig. 6: DigitISA immunosensing of an exosome biomarker.

a Shown is a CD63-specific aptamer bound to CD63 proteins on the surface of the exosomes. The exosome-bound aptamers have a lower electrophoretic mobility than the free aptamers, allowing for the discrimination between exosome-bound and unbound species. b TEM micrographs (left panels) and Western blots (right panels) of exosomes. Exosomes display their characteristic cup-shaped morphology and can be identified by their size (100–200 nm in diameter) and the presence of a lipid bilayer membrane. TEM experiments were repeated three times. Scale bars: 500 nm (top left), 100 nm (bottom left). Western blot (right panels) comparing exosome samples (Exo) and cell lysate sample (CL). CD9 and CD63, both exosome marker proteins, are highly enriched in the exosome sample, while calreticulin (Cal) is excluded from the exosomes, indicating their purity. Western blot experiments were repeated three times. c Electropherograms as obtained by scanning of the confocal volume across the cross-section of the channel in a stepwise manner for the 50 nM exosome–aptamer sample (magenta line; average of N = 3 repeats) and for the free aptamer sample (cyan line; average of N = 3 repeats). The shaded bands correspond to the standard deviation. The light lines with error bands represent the average fluorescence intensity. The solid markers with error bars indicate the number of single-molecule detection events at each coordinate with the error bar representing the standard deviation. The region shaded in grey was used to estimate the concentration of exosomes in the sample. d Exemplary photon-count time traces for the control aptamer-only sample (cyan) and the exosome mixture (magenta) at the position indicated with colored triangles in panel c. Individual exosomes were detected and quantified using a burst-search algorithm as detailed in the Methods section (Data analysis). Dots above the time traces indicate individual exosome detection events. The bin time was 1 ms in all traces. e Mean concentrations of bound exosomes and averaged median binding stoichiometries at three different concentrations of aptamer. Error bars denote the standard deviation of N ≥ 3 repeats. Data points are shown. Source data are provided as a Source Data file.