Droplet-Based Single EV Sequencing for Protein Profiling

The patent uses droplets as intermediary carriers to encapsulate individual EVs along with sequencing reagents. This intermediary approach allows the tiny EVs (below flow cytometry detection limit) to be contained and processed in a larger droplet environment, enabling subsequent molecular amplification and detection that overcomes the size limitation of flow cytometry while maintaining single-EV resolution.

The patent replaces the mechanical/optical detection system of flow cytometry with a molecular biology-based sequencing system. Instead of relying on light scattering and fluorescence detection that fail for sub-micron EVs, the method uses DNA barcoding, PCR amplification, and next-generation sequencing to detect EV proteins, substituting physical measurement with chemical amplification and molecular detection.

The patent segments the bulk EV population into individual single-EV measurements by encapsulating each EV in a separate droplet. This segmentation allows each EV to be independently barcoded and sequenced, preserving the identity and protein composition of rare diseased EV subtypes that would otherwise be masked in bulk measurements, while still enabling high-throughput analysis of thousands of individual EVs.

The patent uses DNA barcodes as information copies of each EV's protein composition. By attaching unique DNA barcodes to antibodies that bind EV proteins, and then amplifying these barcode sequences through PCR and sequencing, the method creates molecular copies that can be detected with high sensitivity, allowing rare EV subtypes to be identified even when present in small numbers within the bulk population.