An antibody-mediated versatile SERS sandwich biosensor for ultrasensitive detection of functional proteins in cells and tissues

Rapid, sensitive, and anti-interference detection of functional proteins, such as G protein-coupled receptors, transporters, and nuclear receptors, holds significant scientific and clinical importance. However, their detection in complex matrices remains challenging for conventional methods like immunoassays, fluorescence, and mass spectrometry, which suffer from limitations in sensitivity, photobleaching, or operational complexity. Surface-enhanced Raman spectroscopy (SERS) has gained popularity as a highly sensitive and versatile platform for such detection. Its performance fundamentally relies on the specificity of molecular recognition and the enhancement efficiency of plasmonic hot spots. Herein, we report a versatile antibody-mediated sandwich immuno-SERS biosensor featuring dual signal enhancement. The antibody-functionalized Fe 3 O 4 @SiO 2 @Ag flower-like hybrid nanostructures and Au@Ag core-shell nanoparticles conjugated with 4-mercaptobenzoic acid as a SERS reporter. Upon target binding, a sandwich immuno-complex is formed, inducing strong plasmonic coupling and resulting in a concentration-dependent SERS signal amplification. The biosensor was thoroughly characterized by scanning electron microscope, transmission electron microscope, dynamic light scattering, ultraviolet-visible, and Raman spectroscopy. Three types of functional proteins, including angiotensin II type 1 receptor (AT 1 R), serotonin transporter (5-HTT), and peroxisome proliferator-activated receptor gamma (PPARγ), in diverse cell lysates and mouse tissues were detected by applying the SERS sensor, with detection limits of 0.26 pg/mL for AT 1 R, 0.31 pg/mL for 5-HTT, and 0.31 pg/mL for PPARγ. The results showed excellent agreement with Western blot analysis, confirming reliability. This work presents a versatile and robust sensing platform for the quantification of low-abundance functional proteins and provides a foundation for future development of point-of-care diagnostics and fundamental biological studies.