Specific isolation and quantification of PD-L1 positive tumor derived exosomes for accurate breast cancer discrimination via aptamer-functionalized magnetic composites and SERS immunoassay.

PD-L1 positive tumor derived exosomes (TEXs^PD-L1) play a significant role in disease progression, tumor metastasis and cancer immunotherapy. However, the overlap of PD-L1 between TEXs and non-tumor derived exosomes (non-TEXs) restricts the specific isolation and quantification of TEX^PD-L1 from clinical samples. Herein, a new aptamer-functionalized and hydrophilic immunomagnetic substrate was designed by decorating generation 5 polyamidoamine dendrimers (G5 PAMAM), zwitterionic trimethylamine N-oxide (TMAO) and EpCAM (Epithelial cell adhesion molecule) aptamers on magnetic cores sequentially (Fe₃O₄@PAMAM@TMAO@Aptamer, named as FPTA) for rapid target and efficient capture of TEXs. The FPTA substrate gathered excellent characters of strong magnetic responsiveness of Fe₃O₄, abundant affinity sites of PAMAM, strong hydrophilicity of TMAO and enhanced affinity properties of EpCAM aptamers. Because of these advantages, FPTA can isolate TEXs quickly within 30min with high capture efficiency of 90.5 % ± 3.0 % and low nonspecific absorption of 8.2 % ± 2.0 % for non-TEXs. Furthermore, PD-L1 (Programmed cell death-ligand 1) positive TEXs (TEXs^PD-L1) from the captured TEXs were recognized and quantitatively analyzed by utilizing SERS (surface-enhanced Raman spectroscopy) reporter molecules 4-NTP (4-Nitrothiophenol) on PD-L1 aptamers-functionalized gold immunoaffinity probe. The signal of TEXs^PD-L1 was converted to SERS signal of 4-NTP at 1344 cm^-1 which exhibited a linear correlation to concentration of TEXs^PD-L1(R² = 0.9905). With these merits, this strategy was further applied to clinical plasma samples from breast cancer (BC) patients and healthy controls (HC), exhibited an excellent diagnosis accuracy with area under curve (AUC) of receiver operating characteristic (ROC) curve reaching 0.988. All these results demonstrate that the FPTA immunomagnetic substrate combined with SERS immunoaffinity probe may become a generic tool for specific isolation and quantitative analysis of PD-L1 positive tumor-derived exosomes in clinics.