Modular secondary-antibody-based SERS immunoprobes for detecting membrane-associated proteins in adherent cells.

Abstract

Cell membrane proteins, including receptors and ion channels, are important biomarkers and drug targets. However, most surface-enhanced Raman scattering (SERS) nanoprobes require direct conjugation of a target-specific antibody, necessitating preparation of a new probe for each target and potentially causing variability in antibody loading and signal output. To address these limitations, we developed a modular SERS immunoassay using goat anti-rabbit immunoglobulin G (GAR) immobilized on Au@Ag nanoparticles (Au@Ag-GAR NPs); targets can be switched by replacing only the rabbit primary antibody. After the primary antibody recognizes its target, nanoparticle-bound GAR binds the primary antibody, linking the SERS nanoprobe to the target and providing a target-associated readout. CD44, a single-pass transmembrane glycoprotein, and Kv1.3, a six-transmembrane-segment voltage-gated potassium channel encoded by KCNA3, were selected to assess applicability across distinct membrane topologies. The Au-core-reporter-Ag-shell architecture generated a stable SERS signal at 1075 cm-1, whereas primary antibodies conferred target specificity. For both recombinant proteins, SERS intensity increased with concentration from 50 to 1000 ng/mL. In 96-well plates, the assay distinguished relative CD44 expression among HEK293T, HCT116, and HeLa cells and Kv1.3 expression between KCNA3-overexpressing and control HEK293T cells, consistent with immunofluorescence and Western blotting. Micro-Raman imaging mapped the spatial distribution of CD44-associated signals in HeLa cells. Thus, the Au@Ag-GAR platform enables plate-based semi-quantitative analysis and spatial Raman imaging of topologically distinct membrane proteins without probe reconstruction for each target.