Intrinsic multi-component fluorescence fingerprint of bovine milk-derived exosomes for label-free biosensing

Bovine milk-derived exosomes are biocompatible nanovesicles with significant potential for drug delivery, yet reliable methods for their label-free identification remain limited. This study employs fluorescence spectroscopy to characterize the intrinsic optical fingerprint of BMEs. Initially, the Excitation-Emission Matrix served as an exploratory tool to map the spectral landscape. Second-derivative analysis was then applied to resolve intrinsic fluorophore hotspots obscured by background interference, followed by spectral deconvolution to separate overlapping components. This analysis revealed a distinct multi-component fluorescence signature arising from Tryptophan, NAD(P)H, AGE-related MRPs, Porphyrins, Flavins (Riboflavin/FAD), Vitamin A, and Schiff-base-type fluorophores. Moreover, all these autofluorescence spectra were recorded at pH 3, 7, and 10, revealing characteristic pH-dependent spectral changes for each component, most notably the emergence of a well-resolved 515 nm peak at pH 3 confirming riboflavin's neutral-to-anionic speciation, as well as a red-shift in porphyrin emission wavelength at pH 3 and 7. Collectively, these intrinsic optical signatures validate BMEs as self-reporting nanocarriers, providing a robust, non-invasive biosensing baseline for quality control and tracking without the structural compromise associated with external tagging.