Finger-prick whole blood analysis via surface-enhanced Raman spectroscopy using centrifugal silver plasmonic paper substrates

Background: Direct surface-enhanced Raman scattering (SERS) analysis of whole blood remains challenging due to spectral interference from hemoglobin, ergothioneine, and other matrix components, typically requiring sample pre-treatment. Here, Centrifugal Silver Plasmonic Paper (CSPP) is applied for the first time to unprocessed capillary blood collected by finger-prick, generating SERS-active silver nanostructures in situ within the same paper strip used for sampling, thereby eliminating pre-treatment and extraction steps. A multi-factor experimental design was used to disentangle four sources of spectral variability (storage temperature, storage time, spatial position along the strip, and inter-donor variability) analysed via the Linear Mixed Model-Principal Components Analysis (LiMM-PCA) framework. Results: All modelled effects were statistically significant (permutation test, p < 0.001). Spatial position along the strip, nested within storage condition, was the dominant source of spectral variance (57%), exceeding inter-donor variability (12%), while storage time and temperature contributed modestly but detectably (<2% each). SERS spectra were dominated by ergothioneine and albumin-bound uric acid bands, with an analyte-specific spatial gradient along the strip consistent with a paper-chromatographic separation mechanism driven by centrifugation. Significance: These findings establish CSPP as a robust, reagent-free platform for direct SERS profiling of metabolites in unprocessed capillary blood. The rigorous variance partitioning provided by LiMM-PCA identifies spatial position as the primary factor governing measurement reproducibility, providing a quantitative basis for protocol optimisation. The approach has direct implications for the development of point-of-care and remote biomonitoring devices.