Cell-Surface-to-Solution Signal Transduction via a CRISPR/Cas12a-Based Dual-Target Aptamer Sensor for Detecting Urothelial Carcinoma Cells in Urine.

Abstract

Urothelial carcinoma (UC) is a common malignancy of the genitourinary system with high recurrence rates, requiring non-invasive postoperative monitoring. Direct detection of exfoliated UC cells in urine can provide tumor-origin evidence but remains challenging because of low target-cell abundance, complex urine interference, and subjective cytological interpretation. Herein, we developed a CRISPR/Cas12a-based dual-target aptamer sensor for sensitive and specific detection of exfoliated UC cells in urine. The sensing system spatially couples CKAP4-mediated magnetic capture with PD-L1-dependent retention of a Cas12a activator on the same captured cell. Magnetic washing removes unbound activator probes, whereas the cell-retained activator subsequently initiates Cas12a trans-cleavage in the solution-phase reaction. This capture-retain-activate architecture provides an AND-gate-like route for converting spatially coupled cell-surface recognition events into an amplified fluorescence output. The sensor achieved a detection limit of 12 cells per assay and showed good selectivity against interfering cells. A pilot clinical evaluation using 32 urine samples distinguished UC patients from non-UC individuals with an area under the receiver operating characteristic curve (AUC) of 0.996. This work provides a capture-retain-activate strategy for the non-invasive analysis of intact exfoliated UC cells and illustrates a potential design route for coupling cell-surface recognition with solution-phase CRISPR/Cas12a signal amplification.