Direct comparison of different protocols to obtain surface enhanced Raman spectra of human serum

Label-free Surface Enhanced Raman Spectroscopy (SERS) is a rapid technique that has been extensively applied in clinical diagnosis and biomedicine for the analysis of biofluids. The purpose of this approach relies on the ability to detect specific "metabolic fingerprints" of complex biological samples, but the full potential of this technique in diagnostics is yet to be exploited, mainly because of the lack of common analytical protocols for sample preparation and analysis. Variation of experimental parameters, such as substrate type, laser wavelength and sample processing can greatly influence spectral patterns, making results from different research groups difficult to compare. This study aims at making a step toward a standardization of the protocols in the analysis of human serum samples with Ag nanoparticles, by directly comparing the SERS spectra obtained from five different methods in which parameters like laser power, nanoparticle concentration, incubation/deproteinization steps and type of substrate used vary. Two protocols are the most used in the literature, and the other three are "in-house" protocols proposed by our group; all of them are employed to analyze the same human serum sample. The experimental results show that all protocols yield spectra that share the same overall spectral pattern, conveying the same biochemical information, but they significantly differ in terms of overall spectral intensity, repeatability, and preparation steps of the sample. A Principal Component Analysis (PCA) was performed revealing that protocol 3 and protocol 1 have the least variability in the dataset, while protocol 2 and 4 are the least repeatable.