Confined Enzyme Probe-Enhanced PER-Cas12a Chemiluminescence Biosensor for Ultrasensitive Detection of Soluble PD-L1 in Serum

Circulating soluble programmed death ligand 1 (sPD-L1), a critical immunomodulatory molecule, holds significant value for early tumor diagnosis, directing immunotherapy, and monitoring prognosis. In this study, we developed an ultra-sensitive and programmable confined enzyme probe-enhanced PER-Cas12a chemiluminescence (CEP-EPC) platform for detecting sPD-L1. This system initiates the trans-cleavage activity of Cas12a via aptamer-targeted recognition of sPD-L1 and a cascade primer exchange reaction (PER). Then, the activated Cas12a cyclically cleaves single-stranded DNA (ssDNA) on the confined enzyme probe, resulting in the release of HRP-labeled ssDNA. Following magnetic separation, the presence of HRP in the homogeneous solution markedly amplifies the chemiluminescence signal in the luminol-hydrogen peroxide (H₂O₂) system. The constructed sensor exhibits a linear range from 5 to 5 × 10³ pg/mL, with a detection limit as low as 1.02 pg/mL. The analytical performance of the developed sensor was validated using 6 cell lines and 112 clinical test samples. Receiver operating characteristic (ROC) curve analysis demonstrated that the strategy could effectively distinguish tumor patients from healthy donors: non-small cell lung cancer (NSCLC, AUC = 0.8750), gastric cancer (AUC = 0.9325), pancreatic cancer (AUC = 0.9725), and breast cancer (AUC = 0.8500). Notably, a significant positive correlation (r = 0.6444, p < 0.0500) was observed between tissue PD-L1 expression and serum sPD-L1 levels in a cohort of 12 NSCLC patients, suggesting the potential application of this strategy in guiding PD-1/PD-L1 immune checkpoint blockade therapies. Collectively, these findings establish the CEP-EPC platform as a robust and promising tool for precision oncology.