Nondestructive separation/enrichment and rolling circle amplification-powered sensitive SERS enumeration of circulating tumor cells via aptamer recognition

Nondestructive separation/enrichment and reliable detection of extremely rare circulating tumor cells (CTCs) in peripheral blood are of considerable importance in tumor precision diagnosis and treatment, yet this remains a big challenge. Herein, a novel strategy for nondestructive separation/enrichment and ultra-sensitive surface-enhanced Raman scattering (SERS)-based enumeration of CTCs is proposed via aptamer recognition and rolling circle amplification (RCA). In this work the magnetic beads modified with "Aptamer (Apt)-Primer" (AP) probes were utilized to specifically capture CTCs, and then after magnetic separation/enrichment, the RCA-powered SERS counting and benzonase nuclease cleavage-assisted nondestructive release of CTCs were realized, respectively. The AP was assembled by hybridizing the EpCAM-specific aptamer with a primer, and the optimal AP contains 4 mismatched bases. The RCA enhanced SERS signal nearly 4.5-fold, and the SERS strategy has good specificity, uniformity and reproducibility. The proposed SERS detection possesses a good linear relationship with the concentration of MCF-7 cells spiked in PBS with the limit of detection (LOD) of 2 cells/mL, which shows good potential practicality for detecting CTCs in blood with recoveries ranging from 100.56% to 116.78%. Besides, the released CTCs remained good cellular activity with the normal proliferation after re-culture for 48 h and normal growth for at least three generations. The proposed strategy of nondestructive separation/enrichment and SERS-based sensitive enumeration is promising for reliable analysis of EpCAM-positive CTCs in blood, which is expected to provide a powerful tool for analysis of extremely rare circulating tumor cells in complex peripheral blood for liquid biopsy.