Hybrid nanoplasmonic porous biomaterial scaffold for liquid biopsy diagnostics using extracellular vesicles

For more effective early-stage cancer diagnostics, there is a need to develop sensitive and specific, non- or minimally invasive, and cost-effective methods for identifying circulating nanoscale extracellular vesicles (EVs). University of California, Davis researchers report the utilization of a simple plasmonic scaffold composed of a microscale biosilicate substrate embedded with silver nanoparticles for surface-enhanced Raman scattering (SERS) analysis of ovarian and endometrial cancer EVs. These substrates are rapidly and inexpensively produced without any complex equipment or lithography. The researchers extensively characterize the substrates with electron microscopy and outline a reproducible methodology for their use in analyzing EVs from in vitro and in vivo biofluids. They report effective chemical treatments for (i) decoration of metal surfaces with cysteamine to nonspecifically pull down EVs to SERS hotspots and (ii) enzymatic cleavage of extraluminal moieties at the surface of EVs that prevent localization of complementary chemical features (lipids/proteins) to the vicinity of the metal-enhanced fields. The researchers observe a major loss of sensitivity for ovarian and endometrial cancer following enzymatic cleavage of EVs’ extraluminal domain, suggesting its critical significance for diagnostic platforms. They  demonstrate that the SERS technique represents an ideal tool to assess and measure the high heterogeneity of EVs isolated from clinical samples in an inexpensive, rapid, and label-free assay.

Overview of the nanoplasmonic substrate and SERS imaging process

(a) Schematic of the SERS optical setup, where the substrate is sandwiched between quartz windows for analysis using an inverted confocal Raman scanning instrument. (b) The biosilicate SERS substrate is irradiated by laser light to instigate Raman scattering. The insets show the heterogeneous surface structure of the compacted diatom mesh at 100× and then under SEM at 10k×, where single diatoms are visible. (c) The substrate allows for transport of EVs from solution to the proximity of AgNP clusters adsorbed to the compacted silicate scaffold. When functionalized with cysteamine, thiol bonds anchor to the AgNPs, enabling anionic EVs to adhere electrostatically to cysteamine’s terminal amine groups. Spectral SERS fingerprints can be acquired from EVs adjacent to AgNPs. (d) SEM micrographs of hybrid biosilicate mesh with AgNP clusters. An Everhart–Thornley detector (ETD) records the secondary electron scattering from the surface, whereas the annular backscattering detector (ABS) collects electrons more sensitive to atomic weight, highlighting the AgNP clusters. The images on the right show likely EV candidates localized in the vicinity of AgNP clusters throughout the hybrid material. The approximate starting concentration of EVs was ∼5 × 10⁸ EV/mL. All scale bars are 1 μm.