Improving spectral reproducibility in SERS analysis of exosomes via surface charge-modulated and biofunctionalized gold nanoparticles

ABSTRACT: This study introduces an improved SERS platform for analyzing exosome heterogeneity. We compared three gold nanoparticle substrates: negatively charged citrate-Au NPs (cit-Au NPs), positively charged cysteamine-Au NPs (cyst-Au NPs), and anti-CD63-functionalized cyst-Au NPs. Conventional cit-/cyst-Au NPs yielded fluctuating SERS signals due to random exosome localization, limiting accurate heterogeneity assessment. In contrast, anti-CD63-functionalized cyst-Au NPs specifically anchored exosomes via CD63 targeting, producing highly reproducible spectra. Principal component analysis (PCA) confirmed significantly reduced spectral variance with the antibody-based approach, with 95% confidence ellipses showing substantially tighter clustering compared to charge-based substrates. Theoretical simulations and sensitivity tests further showed the cyst-Au NPs strategy achieved a detection limit of approximately 1.3×10 3 particles/μL, while cit-Au NPs and anti-CD63-functionalized cyst-Au NPs exhibited LODs of 7.9×10 3 and 8.1×10 4 particles/μL, respectively. Notably, the PCA plot for the biofunctionalized strategy revealed two distinguishable circles, potentially indicating the presence of exosome subpopulations or distinct CD63 epitope distributions. By integrating surface charge control with biofunctionalization, this work enables reproducible, sensitive SERS detection, offering a reliable platform for exosome heterogeneity analysis.