A facile liquid biopsy assay for highly efficient CTCs capture and reagent-less monitoring of immune checkpoint PD-L1 expression on CTCs with non-small cell lung cancer patients.

Programmed cell death 1 ligand 1 (PD-L1) immunotherapy holds a pivotal role in lung cancer treatment. However, current methods for monitoring PD-L1 expression exhibit several limitations, including hysteresis and the invasive nature of tissue sampling. Circulating tumor cells (CTCs), the important biomarkers in liquid biopsy, are minimally invasive and facilitate continuous monitoring. Consequently, integrating CTC counting with PD-L1 expression analysis offers more comprehensive insights for the development of personalized treatment strategies development and efficacy evaluations. In this study, we presented a facile liquid biopsy assay designed for the dynamic monitoring of PD-L1 expression on CTCs captured from non-small cell lung cancer (NSCLC) patients. This assay was achieved by fabricating two high-performance probes: EpCAM and Vimentin dual-aptamer modified nitrogen-doped carbon quantum dots probe (E/V-apt-N-CQDs) and hairpin PD-L1 aptamer coupled with gold nanoparticles (PD-L1-apt-AuNPs). The E/V-apt-N-CQDs probe effectively captured two types of CTC models (H1299 and A549) exhibiting differential PD-L1 expression. Additionally, reagent-less detection of PD-L1 levels on CTCs was achieved using a portable magnetic electrochemical sensor with excellent specificity and sensitivity, which was capable of measuring PD-L1 concentrations as low as 2 ng/mL. Finally, this assay was applied in 41 NSCLC patients to investigate the correlation between CTC numbers or PD-L1 expression and disease progression and immunotherapy efficacy. The results indicated a significant association between elevated CTC counts or reduced PD-L1 levels and clinical progression. Moreover, this liquid assay successfully monitored dynamic changes in CTCs and PD-L1 expression in NSCLC patients receiving immunotherapy, indicating its potential for clinical application.