Fig 2: Anti-CD63 functionalized EPAC specificity.The specificity was studied using EV samples released from SK-MEL-28 and MCF7 cell lines, as well as control experiments including (++) EV-free cell culture medium, (−+) without the CD63 capture antibody, and (+−) with nontarget CD45 detection antibodies on SERS nanotags. (A) The expressions of MCSP, MCAM, ErbB3, and LNGFR in SK-MEL-28 and MCF7 cells were detected by flow cytometry. (B) Representative false-color SERS spectral images, (C) average SERS spectra obtained from corresponding SERS mapping datasets, and (D) average SERS intensities at 1075 cm−1 (red, MCSP), 1375 cm−1 (blue, MCAM), 1335 cm−1 (green, ErbB3), and 1000 cm−1 (yellow, LNGFR). Data in (D) are represented as means ± standard deviation, where error bars represent standard deviation of three separate experiments. Scale bars, 10 μm.

Fig 3: Schematic for EV phenotyping by EPAC.(A) A melanoma cell with a BRAF V600E mutation secretes EVs into circulation or cell culture medium. (B) The sample is directly injected into EPAC, where the applied nanomixing fluid flow increases EV collisions with the capture antibody and SERS nanotags and shears off nontarget molecules (e.g., protein aggregates and apoptotic bodies) and free SERS nanotags. (C) The characterization of EV phenotypes is performed by SERS mapping. The false-color SERS spectral images are established on the basis of the characteristic peak intensities of SERS nanotags (MCSP-MBA, red; MCAM-TFMBA, blue; ErbB3-DTNB, green; LNGFR-MPY, yellow). (D) EV phenotypes defined by the relative expression levels of four biomarkers are extracted from the average signal spectra of false-color SERS spectral images. EV phenotypes are unique to each EV subpopulation. By analyzing EV samples before, during, and after BRAF inhibitor treatment, the phenotypic evolution can be tracked to provide information on treatment responses and early signs of drug resistance.

Fig 4: The anti-MCSP functionalized EPAC for phenotyping of plasma EVs from melanoma patients.(A) EV phenotypes of 15 melanoma samples (P1 to P15) and 12 healthy controls (H1 to H12). P1, P4, P7, and P9 are from the same patient but different time points, as are P5 and P10. (B) Representative false-color SERS spectral images and (C) corresponding average SERS spectra from patient and normal samples (P1, P8, and H1). For (A) and (B), the biomarker signals are represented by red (MCSP), blue (MCAM), green (ErbB3), and yellow (LNGFR). Data in (A) are represented as means ± standard deviation, where error bars represent standard deviation of three separate experiments. Scale bars, 10 μm.