Multivalence-Actuated DNA Nanomachines Enable Bicolor Exosomal Phenotyping and PD-L1-Guided Therapy Monitoring

Exosome-associated liquid biopsies are hampered by challenges in the exosomal quantification and phenotyping. Here, we present a bioinspired exosome-activated DNA molecular machine (ExoADM) with multivalent cyclic amplification that enables highly sensitive detection and phenotyping of circulating exosomes. ExoADM harbors two (an exposed and a hidden) DNA toehold domains that actuate sequential branch migration and multivalent recycling in response to exosomal surface markers. Importantly, this self-powered ExoADM achieves a high sensitivity (33 particles/mu L) and is compatible with another DNA nanomachine targeting different exosomal surface markers for dual-color phenotyping. Using this strategy, we can simultaneously track the dynamic changes of ExoPD-L1 and ExoCD63 expression induced by signaling molecules. Further, we found that their expression levels on circulating exosomes could well differentiate cancer patients from the normal individuals. More importantly, ExoPD-L1 levels could reflect the efficacy of different treatments and guide anti-PD-1 immunotherapy, suggesting the potential of ExoPD-L1 in clinical diagnosis and targeted therapy monitoring.