Raman spectroscopy in extracellular vesicles analysis: Techniques, applications and advancements.

Raman spectroscopy provides a robust approach for detailed analysis of the chemical and molecular profiles of extracellular vesicles (EVs). Recent advancements in Raman techniques have significantly enhanced the sensitivity and accuracy of EV characterization, enabling precise detection and profiling of molecular components within EV samples. This review introduces and compares various Raman-based techniques for EV characterization. These include Raman spectroscopy (RS), which provides fundamental molecular information; Raman trapping analysis (RTA), which combines optical trapping with Raman scattering for the manipulation and analysis of individual EVs; surface-enhanced Raman spectroscopy (SERS), which enhances the Raman signal through the use of metallic nanostructures, significantly improving sensitivity; and microfluidic SERS, which integrates SERS with microfluidic platforms to allow high-throughput, label-free analysis of EVs in biological fluids. In addition to comparing various Raman techniques, this review provides a comprehensive analysis that includes comparisons of machine learning methods, EV isolation techniques, and characterization strategies. By integrating these approaches, the review presents a holistic perspective on Raman-based EV analysis, covering profiling, purity, heterogeneity and size analysis as well as imaging. The combined assessment of Raman technologies with advanced computational and experimental methodologies supports the development of more robust diagnostic and therapeutic applications involving EVs.