Exosomes are secreted nanovesicles which incorporate proteins and nucleic acids, thereby enabling multifunctional pathways for intercellular communication. There is an increasing appreciation of the critical role they play in fundamental processes such as development, wound healing and disease progression, yet because of their heterogeneous molecular content and low concentrations in vivo, their detection and characterization remains a challenge.
In this work researchers from the Naval Research Laboratory combine nano- and microfabrication techniques for the creation of nanosensing arrays tailored toward single exosome detection. Elliptically-shaped nanoplasmonic sensors are fabricated to accommodate at most one exosome and individually imaged in real time, enabling the label-free recording of digital responses in a highly multiplexed geometry. This approach results in a three orders of magnitude sensitivity improvement over previously reported real-time, multiplexed platforms. Each nanosensor is elevated atop a quartz nanopillar, minimizing unwanted nonspecific substrate binding contributions. The approach is validated with the detection of exosomes secreted by MCF7 breast adenocarcinoma cells. The researchers demonstrate the increasingly digital and stochastic nature of the response as the number of subsampled nanosensors is reduced from four hundred to one.
(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.