In situ spatial profiling of cytochrome c release at the single-cell level under photothermal stress utilizing a 3D bifunctional SERS substrate.

Cytochrome c (Cyt c) released from apoptotic cells into extracellular environment during photothermal therapy plays an important role in cell communication, immunological response, acting as a potential tissue messenger. Determining the spatial distribution of Cyt c at a single-cell level is essential to understanding its effects on the surrounding tissue. Here, we report a label-free method for mapping the spatial distribution of Cyt c around single apoptotic cells under photothermal stress using surface-enhanced Raman spectroscopy (SERS). To achieve the in-situ observation of the Cyt c release during photothermal induced apoptosis, we engineer a 3D bifunctional plasmonic nanostructure presenting both optimized photothermal and SERS detection capabilities. The correlation study between the 3D nanostructure and the bifunctional properties is carried out and the measurement results are compared with electrodynamics calculations. Through the 3D bifunctional SERS substrate, we obtain the spatial distribution imaging of Cyt c released by single cells under photothermal stress. To validate the extracellular release pattern of Cyt c, we employ a mitochondrial targeting SERS nanoprobe, allowing for in situ monitoring of the simultaneous release of the nanoprobe and Cyt c during apoptosis. Through this method, we further investigate the variability of extracellular Cyt c release among two distinct cell lines. By developing a 3D bifunctional flexible patch, we achieve in situ spatial mapping of Cyt c released from apoptotic tumor tissues during in vivo photothermal therapy. This approach could deepen insights into photothermal therapy's impact on neighboring cells, potentially optimizing treatments.