Co-profiling of exosomal proteins and microRNAs using single microbead confinement for breast cancer diagnosis

Tumor-derived exosomes (TEXs) hold immense promise as biomarkers for cancer diagnosis, but sensitive and accurate identification is hindered by their intrinsic heterogeneity and extreme scarcity. Co-profiling of exosomal protein and microRNA (miRNA) at the single-molecule level may greatly enhance the precision of cancer diagnosis and prognosis. Herein, we present a single microbead-confined dual-mode detection system that selectively captures TEXs for concurrent quantification of protein and miRNA biomarkers. Exosomal proteins are quantified by measuring fluorescence signal concentrated on a single microbead-based enrichment platform. After on-bead lysis, individual miRNA is recognized by the two peptide nucleic acids (PNAs) to form a strict one-to-one correspondence with the intense light-scattering signal of gold nanoparticles (AuNPs), enabling single-molecule counting analysis. Through comprehensive profiling, a panel of four conjoint biomarkers achieve the highest diagnostic accuracy of 98.33%, outperforming any individual markers or alternative combinations. Importantly, this is the first demonstration of a universal PNA modification strategy that harnesses a DNA mediator to prevent AuNPs self-aggregation. This platform can also be adapted to other cancers by simply changing the aptamers and PNAs to target corresponding biomarkers, showing significant potential for advancing exosome-based liquid biopsies and accelerating the development of next-generation clinical diagnostic tools.