Spatial molecular profiling of living single-cell by membrane-interfaced 3D SERS substrates

Single-cell heterogeneity is a defining feature of biology and medicine, and the cell membrane presents spatially heterogeneous biochemicals that govern cellular interactions. Although fluorescence techniques and Raman spectroscopy have been used to investigate single-cell molecular heterogeneity, label-free, non-destructive acquisition of spatially resolved molecular profiles from living cells remains challenging. Surface-enhanced Raman spectroscopy (SERS) offers high sensitivity and molecular specificity, yet reliable spatial readouts at the single-cell level are limited by the lack of reproducible nano-bio interfaces. Here, we present a membrane-interfaced 3D Au-silica SERS substrate for spatial biochemical profiling of living single cells. By establishing a tight membrane-hotspot interface with uniform SERS performance, the platform supports 2D SERS mapping across individual cell areas and visualizes membrane-associated molecular distributions, revealing biochemical heterogeneity within and between cells. This platform provides a robust basis for precision medicine applications, including cancer subtype identification and monitoring heterogeneous drug responses at the single-cell level.