Applying CRISPR/Cas13 to Construct Exosomal PD‐L1 Ultrasensitive Biosensors for Dynamic Monitoring of Tumor Progression in Immunotherapy

Abstract Programmed cell death receptor 1 (PD‐L1) protein on exosomes (exosomal PD‐L1) is one of the most promising biomarkers for cancer immunotherapy monitoring. However, current approaches for exosomal PD‐L1 detection are poorly sensitive, laborious, and time‐consuming. Here, a new method, named Aptamer‐RPA‐TMA‐Cas13a Assay (ARTCA) is established, which enables exosomal PD‐L1 to be detected directly in serum with a lower limit of 10 particles mL −1 . Mechanistically, using DNA aptamer specifically binding to exosomal PD‐L1, the aptamer is amplified twice by recombinase polymerase amplification (RPA) coupled with transcription‐mediated amplification (TMA) and simultaneously the TMA products are detected in real‐time with CRISPR/Cas13a system. Utilizing ARTCA, PD‐L1 levels in circulating exosomes seem to be a reliable marker of PD‐L1 expression in tumor tissue. The level of circulating exosomal PD‐L1 increases significantly in patients with tumor progression. Ultra‐trace detection of serum exosomal PD‐L1 by ARTCA provides a potentially convenient way for dynamic monitoring of tumor progression for patients undergoing immunotherapy. These results demonstrate the use of CRISPR‐Cas13a for protein detection, and circulating exosomal PD‐L1 levels seem to be a reliable marker as well as PD‐L1 expression in tumor tissue, opening up new avenues for monitoring tumor progression.