Exosomes encapsulate genomic and proteomic biomarkers for non-invasive diagnosis and disease monitoring. However, exosome surface-markers heterogeneity is a major drawback of current isolation methods. Here, we report a direct, one-step exosome sampling technology, ExoPRIME, for selective capture of CD63⁺ exosome subpopulations using an immune-affinity protocol. Microneedles (300μm × 30 mm), functionalized with anti-CD63 antibodies, were incubated under various experimental conditions in conditioned astrocyte medium and astrocyte-derived exosome suspension. The probe's capture efficiency and specificity were validated using FluoroCet assay, immunofluorescent imaging, and OMICS analyses. Significantly higher exosomes were captured by probes incubated for 16 h at 4 ⁰C in enriched exosomal suspension (23 × 10 ⁶ exosomes per probe) vis-à-vis 2 h at 4 0 C (12 × 10 ⁶) and 16 h at 22 ⁰C (3 × 10 ⁶) in conditioned cell media. Our results demonstrate the application of ExoPRIME over a broad dynamic range of temperature and incubation parameters, offering flexibility for any desired application. ExoPRIME permits the use and re-use of minimal sample volumes (≤200 μL), can be multiplexed in arrays, and integrated into a lab-on-a-chip platform to achieve parallel, high-throughput isolation of different exosome classes in a semi-automated workstation. This platform could provide direct exosomal analysis of biological fluids since it can elegantly interface with existing room-temperature, picomolar-range nucleic acid assays to provide a clinical diagnostic tool at the point of care.

Keywords: Exosomes; Immunosensing; Layer-by-layer; Omics; Solid-phase purification.