Exosomes are nanoscale vesicles distinguished by characteristic biophysical and biomolecular features; current analytical approaches, however, remain univariate. Here, researchers from the National University of Singapore develop a dedicated platform for multiparametric exosome analysis—through simultaneous biophysical and biomolecular evaluation of the same vesicles—directly in clinical biofluids. Termed templated plasmonics for exosomes, the technology leverages in situ growth of gold nanoshells on vesicles to achieve multiselectivity. For biophysical selectivity, the nanoshell formation is templated by and tuned to distinguish exosome dimensions. For biomolecular selectivity, the nanoshell plasmonics locally quenches fluorescent probes only if they are target-bound on the same vesicle. The technology thus achieves multiplexed analysis of diverse exosomal biomarkers (e.g., proteins and microRNAs) but remains unresponsive to nonvesicle biomarkers. When implemented on a microfluidic, smartphone-based sensor, the platform is rapid, sensitive, and wash-free. It not only distinguished biomarker organizational states in native clinical samples but also showed that the exosomal subpopulation could more accurately differentiate patient prognosis.
Templated nanoplasmonics for multiparametric profiling of exosomes
(A) Schematic of the TPEX platform. The technology is designed to measure exosomal markers and comprises three functional steps. Exosomes are first labeled with fluorescent molecular probes and AuNP. While AuNP remain well dispersed when associated with nonvesicle, free proteins, they assemble onto exosome periphery, through electrostatic interactions. Excess unbound probes and AuNP are not removed. In the presence of gold salt, the AuNP serve as seeds for in situ gold growth. The dispersed AuNP experience a small growth and a slight shift in their absorbance spectra, leading to minimal changes in the fluorescence signals of probes. The exosome-bound AuNP, on the other hand, develop into a nanoshell; this nanostructure is templated by the vesicle dimension and demonstrates a large red shift in its plasmonic resonance to effectively quench the fluorescence signal of probes bound onto the same vesicle. The TPEX fluorescence signal is thus multiparametric, for both exosomal biophysical characteristics and biomarker compositions. (B) Transmission electron micrographs of TPEX products. In the presence of free proteins, AuNP remained well dispersed (before) and demonstrated a small particle growth after treatment with gold salt (after). When incubated with exosomes, AuNP bound to vesicle periphery (before) and developed into large spherical particles after gold growth (after). Scale bars, 20 nm. (C and D) Photographs of the microfluidic device and the smartphone-based optical detector. Absorbance and fluorescence measurements could be performed on the integrated platform through different light-emitting diode (LED) sources and filter configurations. Scale bar, 1 cm.