Ratiometric SERS platform of dual bioprobes for stable detection of circulating tumor cells

Circulating tumor cells (CTCs) serve as crucial biomarkers for non-invasive cancer diagnostics through liquid biopsy. Surface-enhanced Raman spectroscopy (SERS) bioprobes offer high detection sensitivity of rare CTCs. However, SERS signals often suffer from poor reproducibility due to strong background interference and experimental fluctuations. To address these challenges, we developed a novel ratiometric SERS platform using dual functionalized bioprobes: titanium dioxide nanoparticles conjugated with crystal violet and folic acid (TiO 2 -CV-rBSA-FA), and iron oxide nanoparticles modified with acridine red and an EpCAM antibody (IO–AR–PDA-EpCAM). The ratiometric platform achieves an exceptional limit of detection (LOD) as low as 5 × 10 −7 M. The individual bioprobes demonstrate low signal variability, with relative standard deviation (RSD) values of 7.99 % (TiO₂-CV-rBSA-FA) and 9.00 % (IO-AR-PDA-EpCAM), ensuring consistent performance across repeated measurements. This system operates through coordinated mechanisms where IO-AR-EpCAM bioprobe simultaneously concentrate CTCs through magnetic enrichment while providing AR-based internal calibration signals, whereas TiO 2 -CV-rBSA-FA bioprobe generates CV signals specifically amplified at surfaces of CTCs. The ratiometric measurement (I CV /I AR ) effectively eliminates nonspecific adsorption interference while maintaining excellent reproducibility (relative standard deviation <10 %) and biosafety (cell viability >90 %). This approach established a stable and reliable platform for sensitive CTCs detection.