Aptamer-Coupled Droplet CRISPR/Cas12a Enables Ultrasensitive sPD-L1 Detection

Ultrasensitive detection of soluble programmed death-ligand 1 (sPD-L1) in peripheral blood is essential for early cancer diagnosis and immunotherapy monitoring. Conventional enzyme-linked immunosorbent assays lack the requisite sensitivity, whereas PCR quantifies nucleic-acid surrogates rather than the immunologically active protein. Here we report an aptamer-coupled droplet CRISPR/Cas12a (ADC) platform that integrates a structure-switching aptamer with picolitre droplet microfluidics to achieve femtomolar quantification of sPD-L1 within 70 min. Target binding with aptamer displaces a blocking sequence that activates Cas12a trans-cleavage, generating fluorescent droplets without preamplification. Confinement in picolitre droplets accelerates reaction kinetics through elevated local reagent concentrations and suppresses background fluorescence, collectively enhancing sensitivity. The assay exhibits a 0.5 pM limit of detection for sPD-L1, a dynamic range spanning 3 orders of magnitude, and 100% diagnostic accuracy in blinded plasma from lung cancer patients and healthy donors. The modular ADC architecture is readily adaptable to other protein biomarkers, offering a universal strategy for rapid, ultrasensitive liquid-biopsy analysis.