Reliable Detection of Extracellular PD-L1 by DNA Computation-Mediated Microfluidics

Extracellular vesicle PD-L1 (programmeddeath-1 ligand 1) is ofgreater value in tumor diagnosis, prognosis, and efficacy monitoringof anti-PD-1/PD-L1 immunotherapy. However, soluble PD-L1 interfereswith the accurate detection of extracellular vesicle (EV) PD-L1. Here,we developed a microfluidic differentiationmethod for the detection of extracellular PD-L1,without the interference of soluble, by DNA computation with lipid probes and PD-L1 aptamer as inputs (DECLA). For the developed DECLA method,a cholesterol-DNA probe was designed that efficiently embeds intothe EV membrane, and an aptamer-based PD-L1 probe was used for PD-L1recognition. Due to the stable secondary structure of the designedconnector, only cobinding of cholesterol-DNA and PD-L1 affinity probeinduced biotin-labeled connector activation, while soluble PD-L1 cannothybridize. As a result, PD-L1 EVs can be efficiently captured by streptavidin-functionedherringbone chip and quantified by anti-CD63-induced fluorescencesignal. The high specificity of dual-input DNA computation alliedto the high sensitivity of microfluidic-based detection was suitablefor distinguishing lung cancer patients from healthy donors, highlightingits potential translation to clinical diagnosis and therapy monitoring.