Parallel Analysis of Exosome RNA and Protein for Cancer Diagnosis Using an Integrated on-Chip Microfluidic System

Extracellular vesicle (EVs), particularly exosomes secreted and metabolized by oral tumor cells into saliva, play a role in the tumor metabolism and hold promise as biomarkers for the early detection of oral cancer. In this study, we developed an integrated microfluidic platform for EV processing, enabling the simultaneous analysis of tumor-related exosomal proteins and RNA with high sensitivity on a single chip. The platform utilizes immunomagnetic beads (MBs) for the specific capture of exosomes and integrates two distinct analytical functions. The upper section detects exosomal surface PD-L1 proteins using droplet digital ELISA (ddELISA), while the lower section cleaves the EVs and detects exosomal RNA via Quantitative Real-time polymerase chain reaction (qPCR). This innovative approach eliminates the need for manual liquid transfer between separate off-chip analyses, streamlining the experimental workflow and reducing the risk of sample contamination. Using this platform, we successfully detected PD-L1 positive expressed exosomes with a limit of detection of fewer than 17 exosomes/μL. Additionally, downstream exosome processing, including MBs collection, exosome lysis, RNA reverse transcription, qPCR cycling, and fluorescence analysis, can be completed. These results highlight the platform's potential for early-stage cancer profiling and diagnosis.