Multiplex Quantification of Exosomes via Multiple Types of Nanobeads Labeling Combined with Laser Scanning Detection

In recent years, exosomes have attracted attention in many aspects from basic research to clinical application, including therapeutic reagents or biomarkers for liquid biopsy. The increasing understanding of exosome's heterogeneous properties is expected to lead to more advanced exosome research, and there is therefore a need for a multiplex system that can easily classify and analyze exosomes in complex biological samples according to their properties. In this study, we developed a simple and sensitive multiplexed exosome quantification system based on ExoCounter, an exosome quantification system utilizing optical disk technology, by introducing nanobeads made of different materials as exosome labeling substances. The refractive indices suitable for nanobead materials were analyzed by computer simulation of optical diffraction generated by nanobeads. The results showed that polymer (FG), Au, and Ag nanobeads exhibited superior discrimination capability in terms of the amplitude and polarity of detection pulses generated by each nanobead. The specificity and detection sensitivity of three types of nanobeads were confirmed by detecting HER2-positive exosomes with anti-HER2 antibody-conjugated nanobeads. Furthermore, CD147-positive, HER2-positive, and CD81-positive exosomes in 12.5 μL of serum were simultaneously quantified with high discrimination performance using the anti-CD147 antibody, anti-HER2 antibody, or anti-CD81 antibody conjugated for FG beads, Au nanobeads, or Ag nanobeads, respectively. A limit of detection was also evaluated as low as 210 exosomes/μL. This system is a promising tool for advanced exosome research because it enables multiplexed detection of heterogeneous exosomes in serum with high specificity, accuracy, and sensitivity without purification.