Bioorthogonal Click Chemistry-Enabled Enrichment of Extracellular Vesicles for Integrated Molecular and Functional Liquid Biopsy§.

ConspectusExtracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by virtually all cells, carrying protected lipids, nucleic acids, proteins, and active enzymes that faithfully reflect the physiological and pathological states of their cellular origins. Tumor- and neuron-derived EVs are abundantly present in peripheral blood, even at early disease stages, and thus represent highly attractive substrates for liquid biopsy. However, the clinical translation of EV-based diagnostics has been constrained by a central challenge: the inability to selectively enrich disease-relevant EVs from a vast background of normal EVs with sufficient specificity, efficiency, and compatibility for seamless integration with downstream molecular and functional analyses. Conventional physical isolation approaches generate heterogeneous EV mixtures that dilute disease-specific signals, whereas traditional immunoaffinity capture often suffers from nonspecific interactions and low recovery due to sparse and heterogeneous antigen density on EV membranes.To overcome these limitations, our laboratory has developed a chemical biology solution utilizing the bioorthogonal inverse-electron-demand Diels-Alder reaction between trans-cyclooctene (TCO) and tetrazine (Tz). By labeling tumor or neuronal EVs in plasma with TCO-grafted antibodies and covalently immobilizing them onto Tz-functionalized substrates, our three EV enrichment platforms, namely, EV Click Chips, EV Click Beads, and EV Click MagBeads, enable rapid, irreversible, and highly specific capture of defined EV subpopulations. These click chemistry-mediated enrichment strategies reduce nonspecific binding, markedly improve capture efficiency, and preserve EV integrity, providing a robust foundation for downstream genetic, proteomic, and functional analyses. Building on this chemical biology solution, we established three complementary EV assay modalities. Platform #1, the EV Digital Scoring Assay, couples click chemistry-mediated EV enrichment with RT-digital PCR to quantify tumor-specific mRNAs or oncogenic mutations. This "enrich-then-count" strategy has demonstrated strong clinical utility in early detection of hepatocellular carcinoma (HCC), molecular staging of prostate cancer, and detection of actionable gene alterations in pancreatic cancer and Ewing sarcoma. A refined version enables real-time HCC treatment-response monitoring, outperforming serum AFP and radiographic criteria in monitoring treatment responses. Platform #2, the EV Surface Protein Assay, uses antibody-directed click enrichment followed by immuno-PCR or RT-qPCR to quantify tumor-specific EV subpopulations. Analogous to tissue immunohistochemistry but executed in a liquid-biopsy format, this assay has shown accuracy in early detection of HCC, pancreatic ductal adenocarcinoma, and epithelial ovarian cancer and supports longitudinal monitoring in prostate and thyroid cancers. Platform #3, the EV Protease Activity Assay, extends EV analysis into functional biology by measuring enzymatic activities preserved within enriched EVs. In osteosarcoma, matrix metalloproteinase activity profiles stratified localized versus metastatic disease and tracked therapeutic response. In neurology, quantifying β-secretase activity in neuronal EVs enabled highly accurate detection of early Alzheimer's disease and correlated with cognitive performance.Together, these TCO-Tz click chemistry-enabled platforms provide a modular, robust, and clinically adaptable toolkit for noninvasive EV-based diagnostics. By uniting chemical precision with biological and clinical relevance, this framework advances the broader vision of real-time, disease-specific liquid biopsy across oncology and neurodegeneration, laying the foundation for next-generation integrated diagnostic systems.