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Molecular fingerprints revealed by Raman techniques show great potential for biomedical applications, like disease diagnostic through Raman detection of tumor markers and other molecules in the cell membrane. However, SERS substrates used in membrane molecule studies produce enhanced Raman spectra of high variability and challenging band assignments that limit their application. In this work, these drawbacks are addressed to detect membrane-associated hemoglobin (Hbm) in human erythrocytes through Raman spectroscopy. These cells are incubated with silver nanoparticles (AgNPs) in PBS before Raman measurements. Our results showed that AgNPs form large aggregates in PBS that adhered to the erythrocyte membrane, which enhances Raman scattering by molecules around the membrane, like Hbm. Also, deoxyHb markers may allow Hbm detection in Raman spectra of oxygenated erythrocytes (oxyRBCs). Raman spectra of oxyRBCs incubated with AgNPs showed enhanced deoxyHb signals with good spectral reproducibility, supporting the Hbm detection through deoxyHb markers. Instead, Raman spectra of oxyRBCs showed oxyHb bands associated with free cytoplasmic hemoglobin. Other factors influencing Raman detection of membrane proteins are discussed, like both z-position and dimension of the sample volume. The results encourage membrane protein studies in living cells using Raman spectroscopy, leading to the characterization and diagnostic of different pathologies through a non-invasive technique.

The use of nanodiamonds in studies with living systems generally involves the modification of their surfaces with functional groups. Fluorescent molecules can be attached to these groups, so that one can know the exact position of the particles in each moment of the interaction with the cells. Here we modify the surface of detonation nanodiamonds and nitrogen-vacancy center nanodiamonds using carboxylation and hydroxylation procedures. Subsequent reactions with silicates and cysteine, before addition of fluorescein allow to obtain fluorescent nano-conjugates. We used confocal microscopy to observe the position of nanodiamonds interacting with HeLa cells. At 3 h post-incubation the green fluorescence is localized in extracellular rounded like-vesicles assemblies while at 24 h the conjugates can be observed inside the cells. The measurement of the fluorescence emitted by both conjugates allowed to find an enhanced emission of fluorescein isothiocyanate (FITC) when the nitrogen-vacancy center is present. We propose the existence of a fluorescence enhancement by electron transference process. The procedure described in this work allows the functionalization of nanodiamonds with FITC and other molecules using functional surface groups and small size mediators. Also, as was proved in our work, the nanodiamond-fluorescein conjugates can be used to track nanoparticles position within the cell. Localization studies are particularly important for drug delivery applications of nanodiamonds.