Label-Free Probes and Magneto-Thermal Imprinting Enable Rapid Multiplexed Exosomal Profiling for Breast Cancer Classification

Exosomes, characterized by abundant membrane proteins and high structural stability, have emerged as crucial biomarkers for liquid biopsy. However, conventional exosome isolation and detection methods have limited their clinical applicability due to lengthy procedures, operational complexity, or reliance on chemical labeling. In this study, an integrated platform was developed by combining thermomagnetic molecularly responsive imprinted polymers (TMRMIP) for rapid and selective exosome isolation, with a label-free, electrostatically driven fluorescent aptamer probe for multiplexed detection of exosomal proteins. The TMRMIP features template-matched artificial cavities, enabling direct and efficient capture of target exosomes from complex biological samples, followed by thermally controlled release without additional sample pretreatment. Concurrently, a quaternary ammonium-modified pyrene-based fluorescent probe (TPP) was rationally engineered to electrostatically assemble with aptamers, facilitating ratiometric, array-like fluorescence detection of five representative exosomal membrane proteins (CD63, EpCAM, HER2, CEA, and MUC1), without chemical modification. The platform enabled detection limits down to 1.3 × 10³ particles/mL and supported high-throughput detection in a 96-well format. In a cohort of 60 clinical serum samples, all five protein markers were significantly upregulated in breast cancer patients compared to healthy controls. Linear discriminant analysis (LDA) yielded a diagnostic model with 100% accuracy, sensitivity, and specificity for cancer detection. This work provides a rapid, robust, and scalable label-free approach for exosome profiling, offering strong potential for precise breast cancer diagnostics.