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Molecular diagnostics devoted to discover and monitor new biomarkers is gaining increasing attention in clinical diagnosis. In this work, a programmable DNA-fueled electrochemical analysis strategy is designed for the determination of an emerging biomarker in lung cancer, PD-L1-expressing exosomes. Specifically, PD-L1-expressing exosomes are first enriched onto magnetic beads functionalized with PD-L1 antibody and are able to interact with cholesterol-modified hairpin templates. Then, programmable DNA synthesis starts from the hairpin template-triggered primer exchange reaction and generates a large number of extension products to activate the trans-cleavage activity of CRISPR-Cas12a. After that, CRISPR-Cas12a-catalyzed random cleavage boosts the degradation of methylene blue-labeled signaling strands, so electro-active methylene blue molecules can be enriched onto a cucurbit[7]­uril-modified electrode for quantitative determination. Our method demonstrates high sensitivity and specificity toward electrochemical analysis of PD-L1-expressing exosomes in the range from 103 to 109 particles mL–1 with a low detection limit of 708 particles mL–1. When applied to clinical samples, our method reveals an elevated level of circulating PD-L1-expressing exosomes in lung cancer patients, especially for those at the advanced stages. Therefore, our method may provide new insight into liquid biopsy for better implementation of immunotherapy in lung cancer in the future.

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