Ultrasensitive multiplex SERS immunoassay based on porous Au–Ag alloy nanoparticle–amplified Raman signal probe and encoded photonic crystal beads

An ultrasensitive multiplex surface-enhanced Raman scattering (SERS) immunoassay was developed using porous Au–Ag alloy nanoparticles (p-AuAg NPs) as Raman signal amplification probe coupling with encoded photonic crystal microsphere. p-AuAg NPs were synthesized and modified with the second antibody (Ab₂) and Raman tag (mercaptobenzoic acid, MBA) to prepare a Raman signal–amplified probe. The high porosity of the p-AuAg NPs enables significant coupling of the localized surface plasmon resonance and thus abundant inherent hotspots for Raman signal enhancement. 3D-ordered silver nanoparticles–coated silica photonic crystal beads (Ag/SPCBs) were prepared as encoded SERS substrate for multiplex detection using their reflection peaks. The signal-amplified probe was used for multiplex detection of tumor markers carcinoembryonic antigen (CEA) and alpha fetoprotein (AFP). The wide linear ranges of 10⁻⁷–10³ ng/mL for CEA and 10⁻⁴–10³ ng/mL for AFP with detection limits of 1.22 × 10⁻⁸ ng/mL and 2.47 × 10⁻⁵ ng/mL for CEA and AFP at a signal-to-noise ratio of 3 were obtained. The proposed multiplex SERS immunoassay method displays ultrahigh sensitivity, wide linear range, and excellent specificity, which can be successfully applied to measure clinical serum samples with satisfactory results. The research provides a novel SERS signal enhancement strategy for the multiplex bioassay. Graphical