Fig 1.
(A) 25.4 mm diameter LSPRi sensor chip. (B) LSPRi image of a 20 × 20 array, with a pitch size of 600 nm scale bar: 1 μm. (C) LSPRi image of sixteen arrays in the FOV taken using 100X / 1.4 NA objective, each consisting of 400 plasmonic nanopillars in a 20 × 20 square lattice and 500 nm pitch, scale bar: 10 μm. (D) False colored SEM image of a 10 × 10 nanopillar array, scale bar: 1 μm. (E) High-magnification false colored SEM image showing detailed view of individual nanopillars, scale bar: 200 nm. (F) Diagram illustrating size matching of individual nanopillars diameter (d = 90 nm) to that of exosomes (~50 nm < d < 200 nm), allowing digitized exosome detection while also elevating the sensor to minimize background contributions from the substrate.
Fig 2.
MCF7 exosome characterization.
(A) Western blot analysis of exosomes (i) MCF7 exosome protein profile (ii) Colorimetric image (CA) lane displays standard weight markers and chemiluminescent image (CL) lane shows CD63 detection near 55 kD. (iii) Colorimetric image (CA) lane displays standard weight markers and chemiluminescent image (CL) lane shows CD9 detection near 28 kD. (B) Nanosight analysis of MCF7 exosomes showing the size distribution. (C) AFM image of a single MCF7 exosomes in 0.1 M PBS on freshly cleaved mica. (D) AFM image of larger populations of MCF7 exosomes. (E) Representative cross-sections of several exosomes taken from AFM scans.
Fig 3.
Spectral LSPR data versus LSPRi.
(A) LSPR spectra from a 400 nanopillar array taken before (black) and after (red) introducing the exosomes (105 exosomes/ml). The spectrometer’s spatial resolution and integration time were 200 μm2 and 5 sec, respectively (B) CMOS Image of the same array allowing for single nanopillar resolution, 0.5 μm2 resolution, and a faster integration time of 1.3 sec. Grid overlay denotes region of interest for individual nanopillar intensity measurements. In LSPRi the nanopillars brighten in response to analyte as a result of the shifting spectra. Scale bar = 1 um.
Fig 4.
Subsampled LSPRi time series from a 400 nanopillar array (background subtracted).
The blue, green and red squares on the inset images encompass the subsampled nanopillars, the average response of which is plotted in the corresponding color. For all panels, 1 × 105 exosomes/ml was introduced at t ~ 1200 sec. (A) Spatially averaged time course of the entire 20 × 20 array (144 μm2) in blue. (B) Spatially averaged time course of the entire 20 × 20 array (144 μm2) and the 4 × 4 subarray (5.8 μm2) outlined in green (C) Spatially averaged time course of the entire 20 × 20 array (144 μm2), the 4 × 4 subarray (5.8 μm2) outlined in green and the 4 × 4 subarray (5.8 μm2) outlined in red. (D) Spatially averaged time course of the entire 20 × 20 array (144 μm2) and a separate control experiment (black data) on the same array functionalized with the IgG control antibodies.
Fig 5.
(A) Spatially averaged data from three individual nanopillars (0.36 μm2) in blue, red, and black, respectively (background subtracted). (B) LSPRi of 20 x 20 nanosensor array highlighting a subsampled area for individual nanostructure measurements. Scale bar 1 μm. (C) Zoomed in view of the single nanosensor measurement area. 1 × 105 exosomes/ml were introduced at t ~ 1200 sec. The sharp jumps and stochastic nature of the detection events are consistent with single exosome detection.
Fig 6.
LSPRi versus SPRi exosome detection (background subtracted).
LSPRi data (orange line) are from the green 4 × 4 subarray (5.8 μm2) of Fig 4 while the SPRi data (red line) are from a single sensor of the Biorad XPR36 SPRi system (250,000 μm2). In both experiments, an exosome concentration of 1 × 105 exosomes/ml was introduced. The data sets have been offset in time so that the introductions of analyte coincide.